https://bradleymonk.com/wiki/api.php?action=feedcontributions&feedformat=atom&user=Monakhosbradwiki - User contributions [en]2026-04-09T17:40:00ZUser contributionsMediaWiki 1.41.0https://bradleymonk.com/wiki/index.php?title=Neural_Nets&diff=3922Neural Nets2020-05-04T12:20:15Z<p>Monakhos: /* Tensorflow Playground */</p>
<hr />
<div>{{SmallBox|float=right|clear=none|margin=0px 0px 8px 18px|width=170px|font-size=13px|Tutorial Pages|txt-size=11px|<br />
1. [[Neural Nets|Intro]]<br><br />
2. [[Neural Nets 2|Network Inputs]]<br><br />
3. [[Neural Nets 3|Network Activation]]<br><br />
4. [[Neural Nets 4|Network Outputs]]<br><br />
}}<br />
Below there is an embedded a neural network classifier, rendered using [https://www.tensorflow.org Tensorflow][http://playground.tensorflow.org Playground]. There are a variety of knobs and buttons on the interface; as we move along, more of these options will become available. Don't worry though, all these will be explained in detail, in due time. For now though, let's define our primary goal throughout this tutorial: '''classification'''.<br />
<br />
The primary task is to train neural nets to classify items into categories, based on some limited information. Like fruit or vegetable; or undergrad major; or Alzheimer's Disease patient (CASE) or control participant (CTRL). In the Tensorflow playground below, you can see a bunch of orange and blue dots. Instead of simply thinking about these as dots at arbitrary spatial coordinates, it will be helpful to think of these as representing people in a clinical study. Let's define the blue dots are patients from the CASE group, and orange dots are CTRL participants. What is our 'limited information' about them? Let's say we have collected information about their age and their score on a dementia screening exam (scores represent number of items forgotten). So the first thing we'd probably want to do is make a scatter plot of these two variables. Let's define their respective dimensions on the plot axes as:<br />
<br />
* x-axis | dim1 | current age ('''''AGE''''')<br />
* y-axis | dim2 | exam score ('''''SCORE''''')<br />
<br />
Notice that after plotting, the dots seem to form clusters. That very promising! If you were asked to draw a line on this plane, to separate these two clusters, it could be easily done. Our brain's neural nets have already solved the the spatial problem. Now let's see if an artificial neural net can solve the same problem. <br />
<br />
Click the blue ''start'' button below; let it run for about 500 epochs (~5 seconds), then click pause.<br />
<br />
{{Clear}}<br />
<br />
===Tensorflow Playground===<br />
<br />
<iframe key="tf" path="#activation=tanh&regularization=L1&batchSize=15&dataset=gauss&regDataset=reg-plane&learningRate=0.003&regularizationRate=0.001&noise=0&networkShape=1&seed=0.73576&showTestData=false&discretize=false&percTrainData=40&x=true&y=false&xTimesY=false&xSquared=false&ySquared=false&cosX=false&sinX=false&cosY=false&sinY=false&collectStats=false&problem=classification&initZero=false&hideText=false&showTestData_hide=true&stepButton_hide=true&problem_hide=true&noise_hide=true&discretize_hide=true&regularization_hide=true&dataset_hide=true&batchSize_hide=true&percTrainData_hide=true&regularizationRate_hide=true&learningRate_hide=true&numHiddenLayers_hide=true" /><br />
<br />
<br />
<br />
<br />
{{Clear}}<br />
<br />
Is one neuron with input from ''a single feature'' (the dim1 data: AGE) performing well in the separation task? If so, an orange-colored background should have formed behind the orange dots, while a blue-colored background should have formed behind the blue dots. This colored surface gradient can be understood as the neural network's prediction value at that given coordinate. We will explore prediction values in more detail later on in the tutorial. First let's take a look at what the neural net is taking as inputs.<br />
<br />
{{Clear}}<br />
<br />
===Inputs===<br />
----<br />
{{SmallBox|display=block <br />
|float=right <br />
|clear=none <br />
|width=250px<br />
|margin=5px 2%<br />
|border-width=2px <br />
|border-radius=2px<br />
|[[File: Neural Net Features.png|250px]]<br />
| Figure 1<br />
}}<br />
<br />
To assess the performance, take a look at the input options in ''Figure-1''. There are a bunch of ''X'' variables with subscripts and superscripts, and next to each is a box with various color gradients. For now, let's focus on just two of those symbols, and what they mean to us...<br />
<br />
{| class="wikitable" width=30% align=center <br />
|+ style="font-weight:bold;"|Input Features<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<br />
|}<br />
<br />
These are parsed such that subscripts (''X''<sub>1</sub> , ''X''<sub>2</sub> ,... ''X''<sub>i</sub> ) represent each predictor variable, like AGE and SCORE. As you can see, the first two input options ''X''<sub>1</sub> and ''X''<sub>2</sub> are just ''X''<sub>AGE</sub> and ''X''<sub>SCORE</sub>. Note that since ''X''<sub>1</sub> is plotted on the x-axis, it has a color gradient that changes horizontally, but is constant in the vertical dimension. Conversely the ''X''<sub>2</sub> feature plotted on the y-axis has a vertical color gradient. To clarify why this happens...<br />
<br />
If the only thing we know about these study participants is their AGE, ''X''<sub>1</sub>, we can only make a 1-D plot with each person's age along the x-axis, such that [ x = ''AGE''<sub>i</sub> , y = 0 ]. If you take a look at Figure-2, it should be clear that when information is collapsed onto its single dimension and plotted along the x-axis, the best line we can draw to separate the dim-1 data will be orthogonal to the x-axis (a vertical line). As you move horizontally along the x-axis your categorical guess will likely change, along with the confidence in that guess, which is precisely what is being represented by the color gradient. On the other hand, knowing nothing about exam score, moving up and down on the y-axis will have no effect on your decision, which is why color is constant in the y-dimension. <br />
<br />
When the neural net only gets input about a single feature of each person in the dataset, its synaptic weights will only adapt output along that one dimension. Thus, if for example the network sees that a person is 3 years above the dataset average (considering the data has been ''mean deviated'' and centered), it won't matter what that person's cognitive SCORE was (since the neural net doesn't have access to that info), the network will always make the same guess for anyone 3 years above average age. This is why color is constant at ''x''=3 for any ''y'' value.<br />
<br />
{{Clear}}<br />
<br />
{{SmallBox|display=block <br />
|float=left <br />
|clear=none <br />
|width=420px<br />
|margin=15px 5%<br />
|border-width=2px <br />
|border-radius=2px<br />
|[[File:NN NumberLine.png|400px]]<br />
| Figure 2<br />
}}<br />
<br />
<br />
This isn't a shortcoming of having just one single neuron in the entire network. You could add as many neurons and layers as you want (go ahead and try it)...... if the network only gets input about one feature dimension, the output will be the same, whether there is 1 neuron, or 1 billion. To realize this fact, pretend you can only see the dots as they are plotted in along the number line in 1D (in Figure 2); if we were unable to see the 2D cluster clouds above that line, the billions of neurons in our brain would tell us to draw the classification line in basically the same place as that one single neuron in our artificial neural net. This is a very interesting concept worth noting: neural net classifiers can fail for two very different reasons.<br />
<br />
(1) The neural network itself might be ill-formulated in such a way that, no matter how much information you provide, it cannot seem to learn to solve the classification problem. (2) On the other hand, you might have implemented an apposite deep neural network; yet if the input data is insufficient to solve the classification problem, it will appear to you that this potentially very good neural network performs like garbage. <br><br />
<br />
With that said, there are ways to help prevent that later scenario from happening. These involve doing things like you see for the rest of the input features. The next page will discuss the full set of possible network inputs we have here, which includes...<br />
<br />
<br />
{{Clear}}<br />
<br />
{| class="wikitable" width=30% align=center <br />
|+ style="font-weight:bold;"|Input Features<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub><sup>2</sup> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<sup>2</sup><br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub><sup>2</sup> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<sup>2</sup><br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub>''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE × SCORE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| sin(''X''<sub>1</sub>) <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| sin(AGE)<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| sin(''X''<sub>2</sub>) <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| sin(SCORE)<br />
|}<br />
<br />
<br />
{{Clear}}<br />
{{SmallBox|'''[[Neural Nets 2|Continue to Neural Nets Tutorial Page 2]]'''}}<br />
<br />
<!-- <btn data-toggle="tooltip">Neural Nets 2</btn> --></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Neural_Nets&diff=3921Neural Nets2020-05-04T11:52:29Z<p>Monakhos: </p>
<hr />
<div>{{SmallBox|float=right|clear=none|margin=0px 0px 8px 18px|width=170px|font-size=13px|Tutorial Pages|txt-size=11px|<br />
1. [[Neural Nets|Intro]]<br><br />
2. [[Neural Nets 2|Network Inputs]]<br><br />
3. [[Neural Nets 3|Network Activation]]<br><br />
4. [[Neural Nets 4|Network Outputs]]<br><br />
}}<br />
Below there is an embedded a neural network classifier, rendered using [https://www.tensorflow.org Tensorflow][http://playground.tensorflow.org Playground]. There are a variety of knobs and buttons on the interface; as we move along, more of these options will become available. Don't worry though, all these will be explained in detail, in due time. For now though, let's define our primary goal throughout this tutorial: '''classification'''.<br />
<br />
The primary task is to train neural nets to classify items into categories, based on some limited information. Like fruit or vegetable; or undergrad major; or Alzheimer's Disease patient (CASE) or control participant (CTRL). In the Tensorflow playground below, you can see a bunch of orange and blue dots. Instead of simply thinking about these as dots at arbitrary spatial coordinates, it will be helpful to think of these as representing people in a clinical study. Let's define the blue dots are patients from the CASE group, and orange dots are CTRL participants. What is our 'limited information' about them? Let's say we have collected information about their age and their score on a dementia screening exam (scores represent number of items forgotten). So the first thing we'd probably want to do is make a scatter plot of these two variables. Let's define their respective dimensions on the plot axes as:<br />
<br />
* x-axis | dim1 | current age ('''''AGE''''')<br />
* y-axis | dim2 | exam score ('''''SCORE''''')<br />
<br />
Notice that after plotting, the dots seem to form clusters. That very promising! If you were asked to draw a line on this plane, to separate these two clusters, it could be easily done. Our brain's neural nets have already solved the the spatial problem. Now let's see if an artificial neural net can solve the same problem. <br />
<br />
Click the blue ''start'' button below; let it run for about 500 epochs (~5 seconds), then click pause.<br />
<br />
{{Clear}}<br />
<br />
===Tensorflow Playground===<br />
<br />
<iframe key="tf" path="#activation=tanh&regularization=L1&batchSize=15&dataset=gauss&regDataset=reg-plane&learningRate=0.003&regularizationRate=0.001&noise=0&networkShape=1&seed=0.73576&showTestData=false&discretize=false&percTrainData=40&x=true&y=false&xTimesY=false&xSquared=false&ySquared=false&cosX=false&sinX=false&cosY=false&sinY=false&collectStats=false&problem=classification&initZero=false&hideText=false&showTestData_hide=true&stepButton_hide=true&problem_hide=true&noise_hide=true&discretize_hide=true&regularization_hide=true&dataset_hide=true&batchSize_hide=true&percTrainData_hide=true&regularizationRate_hide=true&learningRate_hide=true&numHiddenLayers_hide=true" /><br />
<br />
<br />
<!--<br />
<html><br />
<iframe src="http://playground.tensorflow.org/#activation=tanh&regularization=L1&batchSize=15&dataset=gauss&regDataset=reg-plane&learningRate=0.003&regularizationRate=0.001&noise=0&networkShape=1&seed=0.73576&showTestData=false&discretize=false&percTrainData=40&x=true&y=false&xTimesY=false&xSquared=false&ySquared=false&cosX=false&sinX=false&cosY=false&sinY=false&collectStats=false&problem=classification&initZero=false&hideText=false&showTestData_hide=true&stepButton_hide=true&problem_hide=true&noise_hide=true&discretize_hide=true&regularization_hide=true&dataset_hide=true&batchSize_hide=true&percTrainData_hide=true&regularizationRate_hide=true&learningRate_hide=true&numHiddenLayers_hide=true" height="1050" width="1050" scrolling="no" style="border:none; overflow:hidden; padding-top:-220px; margin-top:-220px;"></iframe> <br />
</html><br />
--><br />
<br />
{{Clear}}<br />
<br />
Is one neuron with input from ''a single feature'' (the dim1 data: AGE) performing well in the separation task? If so, an orange-colored background should have formed behind the orange dots, while a blue-colored background should have formed behind the blue dots. This colored surface gradient can be understood as the neural network's prediction value at that given coordinate. We will explore prediction values in more detail later on in the tutorial. First let's take a look at what the neural net is taking as inputs.<br />
<br />
{{Clear}}<br />
<br />
===Inputs===<br />
----<br />
{{SmallBox|display=block <br />
|float=right <br />
|clear=none <br />
|width=250px<br />
|margin=5px 2%<br />
|border-width=2px <br />
|border-radius=2px<br />
|[[File: Neural Net Features.png|250px]]<br />
| Figure 1<br />
}}<br />
<br />
To assess the performance, take a look at the input options in ''Figure-1''. There are a bunch of ''X'' variables with subscripts and superscripts, and next to each is a box with various color gradients. For now, let's focus on just two of those symbols, and what they mean to us...<br />
<br />
{| class="wikitable" width=30% align=center <br />
|+ style="font-weight:bold;"|Input Features<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<br />
|}<br />
<br />
These are parsed such that subscripts (''X''<sub>1</sub> , ''X''<sub>2</sub> ,... ''X''<sub>i</sub> ) represent each predictor variable, like AGE and SCORE. As you can see, the first two input options ''X''<sub>1</sub> and ''X''<sub>2</sub> are just ''X''<sub>AGE</sub> and ''X''<sub>SCORE</sub>. Note that since ''X''<sub>1</sub> is plotted on the x-axis, it has a color gradient that changes horizontally, but is constant in the vertical dimension. Conversely the ''X''<sub>2</sub> feature plotted on the y-axis has a vertical color gradient. To clarify why this happens...<br />
<br />
If the only thing we know about these study participants is their AGE, ''X''<sub>1</sub>, we can only make a 1-D plot with each person's age along the x-axis, such that [ x = ''AGE''<sub>i</sub> , y = 0 ]. If you take a look at Figure-2, it should be clear that when information is collapsed onto its single dimension and plotted along the x-axis, the best line we can draw to separate the dim-1 data will be orthogonal to the x-axis (a vertical line). As you move horizontally along the x-axis your categorical guess will likely change, along with the confidence in that guess, which is precisely what is being represented by the color gradient. On the other hand, knowing nothing about exam score, moving up and down on the y-axis will have no effect on your decision, which is why color is constant in the y-dimension. <br />
<br />
When the neural net only gets input about a single feature of each person in the dataset, its synaptic weights will only adapt output along that one dimension. Thus, if for example the network sees that a person is 3 years above the dataset average (considering the data has been ''mean deviated'' and centered), it won't matter what that person's cognitive SCORE was (since the neural net doesn't have access to that info), the network will always make the same guess for anyone 3 years above average age. This is why color is constant at ''x''=3 for any ''y'' value.<br />
<br />
{{Clear}}<br />
<br />
{{SmallBox|display=block <br />
|float=left <br />
|clear=none <br />
|width=420px<br />
|margin=15px 5%<br />
|border-width=2px <br />
|border-radius=2px<br />
|[[File:NN NumberLine.png|400px]]<br />
| Figure 2<br />
}}<br />
<br />
<br />
This isn't a shortcoming of having just one single neuron in the entire network. You could add as many neurons and layers as you want (go ahead and try it)...... if the network only gets input about one feature dimension, the output will be the same, whether there is 1 neuron, or 1 billion. To realize this fact, pretend you can only see the dots as they are plotted in along the number line in 1D (in Figure 2); if we were unable to see the 2D cluster clouds above that line, the billions of neurons in our brain would tell us to draw the classification line in basically the same place as that one single neuron in our artificial neural net. This is a very interesting concept worth noting: neural net classifiers can fail for two very different reasons.<br />
<br />
(1) The neural network itself might be ill-formulated in such a way that, no matter how much information you provide, it cannot seem to learn to solve the classification problem. (2) On the other hand, you might have implemented an apposite deep neural network; yet if the input data is insufficient to solve the classification problem, it will appear to you that this potentially very good neural network performs like garbage. <br><br />
<br />
With that said, there are ways to help prevent that later scenario from happening. These involve doing things like you see for the rest of the input features. The next page will discuss the full set of possible network inputs we have here, which includes...<br />
<br />
<br />
{{Clear}}<br />
<br />
{| class="wikitable" width=30% align=center <br />
|+ style="font-weight:bold;"|Input Features<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub><sup>2</sup> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<sup>2</sup><br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub><sup>2</sup> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<sup>2</sup><br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub>''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE × SCORE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| sin(''X''<sub>1</sub>) <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| sin(AGE)<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| sin(''X''<sub>2</sub>) <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| sin(SCORE)<br />
|}<br />
<br />
<br />
{{Clear}}<br />
{{SmallBox|'''[[Neural Nets 2|Continue to Neural Nets Tutorial Page 2]]'''}}<br />
<br />
<!-- <btn data-toggle="tooltip">Neural Nets 2</btn> --></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Neural_Nets&diff=3920Neural Nets2020-05-04T11:41:21Z<p>Monakhos: </p>
<hr />
<div>{{SmallBox|float=right|clear=none|margin=0px 0px 8px 18px|width=170px|font-size=13px|Tutorial Pages|txt-size=11px|<br />
1. [[Neural Nets|Intro]]<br><br />
2. [[Neural Nets 2|Network Inputs]]<br><br />
3. [[Neural Nets 3|Network Activation]]<br><br />
4. [[Neural Nets 4|Network Outputs]]<br><br />
}}<br />
Below there is an embedded a neural network classifier, rendered using [https://www.tensorflow.org Tensorflow][http://playground.tensorflow.org Playground]. There are a variety of knobs and buttons on the interface; as we move along, more of these options will become available. Don't worry though, all these will be explained in detail, in due time. For now though, let's define our primary goal throughout this tutorial: '''classification'''.<br />
<br />
The primary task is to train neural nets to classify items into categories, based on some limited information. Like fruit or vegetable; or undergrad major; or Alzheimer's Disease patient (CASE) or control participant (CTRL). In the Tensorflow playground below, you can see a bunch of orange and blue dots. Instead of simply thinking about these as dots at arbitrary spatial coordinates, it will be helpful to think of these as representing people in a clinical study. Let's define the blue dots are patients from the CASE group, and orange dots are CTRL participants. What is our 'limited information' about them? Let's say we have collected information about their age and their score on a dementia screening exam (scores represent number of items forgotten). So the first thing we'd probably want to do is make a scatter plot of these two variables. Let's define their respective dimensions on the plot axes as:<br />
<br />
* x-axis | dim1 | current age ('''''AGE''''')<br />
* y-axis | dim2 | exam score ('''''SCORE''''')<br />
<br />
Notice that after plotting, the dots seem to form clusters. That very promising! If you were asked to draw a line on this plane, to separate these two clusters, it could be easily done. Our brain's neural nets have already solved the the spatial problem. Now let's see if an artificial neural net can solve the same problem. <br />
<br />
Click the blue ''start'' button below; let it run for about 500 epochs (~5 seconds), then click pause.<br />
<br />
{{Clear}}<br />
<br />
===Tensorflow Playground===<br />
<html><br />
<iframe src="http://bradleymonk.com"></iframe> <br />
</html><br />
<br />
<html><br />
<iframe src="http://playground.tensorflow.org/#activation=tanh&regularization=L1&batchSize=15&dataset=gauss&regDataset=reg-plane&learningRate=0.003&regularizationRate=0.001&noise=0&networkShape=1&seed=0.73576&showTestData=false&discretize=false&percTrainData=40&x=true&y=false&xTimesY=false&xSquared=false&ySquared=false&cosX=false&sinX=false&cosY=false&sinY=false&collectStats=false&problem=classification&initZero=false&hideText=false&showTestData_hide=true&stepButton_hide=true&problem_hide=true&noise_hide=true&discretize_hide=true&regularization_hide=true&dataset_hide=true&batchSize_hide=true&percTrainData_hide=true&regularizationRate_hide=true&learningRate_hide=true&numHiddenLayers_hide=true" height="1050" width="1050" scrolling="no" style="border:none; overflow:hidden; padding-top:-220px; margin-top:-220px;"></iframe> <br />
</html><br />
<br />
{{Clear}}<br />
<br />
Is one neuron with input from ''a single feature'' (the dim1 data: AGE) performing well in the separation task? If so, an orange-colored background should have formed behind the orange dots, while a blue-colored background should have formed behind the blue dots. This colored surface gradient can be understood as the neural network's prediction value at that given coordinate. We will explore prediction values in more detail later on in the tutorial. First let's take a look at what the neural net is taking as inputs.<br />
<br />
{{Clear}}<br />
<br />
===Inputs===<br />
----<br />
{{SmallBox|display=block <br />
|float=right <br />
|clear=none <br />
|width=250px<br />
|margin=5px 2%<br />
|border-width=2px <br />
|border-radius=2px<br />
|[[File: Neural Net Features.png|250px]]<br />
| Figure 1<br />
}}<br />
<br />
To assess the performance, take a look at the input options in ''Figure-1''. There are a bunch of ''X'' variables with subscripts and superscripts, and next to each is a box with various color gradients. For now, let's focus on just two of those symbols, and what they mean to us...<br />
<br />
{| class="wikitable" width=30% align=center <br />
|+ style="font-weight:bold;"|Input Features<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<br />
|}<br />
<br />
These are parsed such that subscripts (''X''<sub>1</sub> , ''X''<sub>2</sub> ,... ''X''<sub>i</sub> ) represent each predictor variable, like AGE and SCORE. As you can see, the first two input options ''X''<sub>1</sub> and ''X''<sub>2</sub> are just ''X''<sub>AGE</sub> and ''X''<sub>SCORE</sub>. Note that since ''X''<sub>1</sub> is plotted on the x-axis, it has a color gradient that changes horizontally, but is constant in the vertical dimension. Conversely the ''X''<sub>2</sub> feature plotted on the y-axis has a vertical color gradient. To clarify why this happens...<br />
<br />
If the only thing we know about these study participants is their AGE, ''X''<sub>1</sub>, we can only make a 1-D plot with each person's age along the x-axis, such that [ x = ''AGE''<sub>i</sub> , y = 0 ]. If you take a look at Figure-2, it should be clear that when information is collapsed onto its single dimension and plotted along the x-axis, the best line we can draw to separate the dim-1 data will be orthogonal to the x-axis (a vertical line). As you move horizontally along the x-axis your categorical guess will likely change, along with the confidence in that guess, which is precisely what is being represented by the color gradient. On the other hand, knowing nothing about exam score, moving up and down on the y-axis will have no effect on your decision, which is why color is constant in the y-dimension. <br />
<br />
When the neural net only gets input about a single feature of each person in the dataset, its synaptic weights will only adapt output along that one dimension. Thus, if for example the network sees that a person is 3 years above the dataset average (considering the data has been ''mean deviated'' and centered), it won't matter what that person's cognitive SCORE was (since the neural net doesn't have access to that info), the network will always make the same guess for anyone 3 years above average age. This is why color is constant at ''x''=3 for any ''y'' value.<br />
<br />
{{Clear}}<br />
<br />
{{SmallBox|display=block <br />
|float=left <br />
|clear=none <br />
|width=420px<br />
|margin=15px 5%<br />
|border-width=2px <br />
|border-radius=2px<br />
|[[File:NN NumberLine.png|400px]]<br />
| Figure 2<br />
}}<br />
<br />
<br />
This isn't a shortcoming of having just one single neuron in the entire network. You could add as many neurons and layers as you want (go ahead and try it)...... if the network only gets input about one feature dimension, the output will be the same, whether there is 1 neuron, or 1 billion. To realize this fact, pretend you can only see the dots as they are plotted in along the number line in 1D (in Figure 2); if we were unable to see the 2D cluster clouds above that line, the billions of neurons in our brain would tell us to draw the classification line in basically the same place as that one single neuron in our artificial neural net. This is a very interesting concept worth noting: neural net classifiers can fail for two very different reasons.<br />
<br />
(1) The neural network itself might be ill-formulated in such a way that, no matter how much information you provide, it cannot seem to learn to solve the classification problem. (2) On the other hand, you might have implemented an apposite deep neural network; yet if the input data is insufficient to solve the classification problem, it will appear to you that this potentially very good neural network performs like garbage. <br><br />
<br />
With that said, there are ways to help prevent that later scenario from happening. These involve doing things like you see for the rest of the input features. The next page will discuss the full set of possible network inputs we have here, which includes...<br />
<br />
<br />
{{Clear}}<br />
<br />
{| class="wikitable" width=30% align=center <br />
|+ style="font-weight:bold;"|Input Features<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub><sup>2</sup> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<sup>2</sup><br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub><sup>2</sup> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<sup>2</sup><br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub>''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE × SCORE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| sin(''X''<sub>1</sub>) <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| sin(AGE)<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| sin(''X''<sub>2</sub>) <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| sin(SCORE)<br />
|}<br />
<br />
<br />
{{Clear}}<br />
{{SmallBox|'''[[Neural Nets 2|Continue to Neural Nets Tutorial Page 2]]'''}}<br />
<br />
<!-- <btn data-toggle="tooltip">Neural Nets 2</btn> --></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Neural_Nets&diff=3919Neural Nets2020-05-04T11:38:34Z<p>Monakhos: /* Tensorflow Playground */</p>
<hr />
<div>{{SmallBox|float=right|clear=none|margin=0px 0px 8px 18px|width=170px|font-size=13px|Tutorial Pages|txt-size=11px|<br />
1. [[Neural Nets|Intro]]<br><br />
2. [[Neural Nets 2|Network Inputs]]<br><br />
3. [[Neural Nets 3|Network Activation]]<br><br />
4. [[Neural Nets 4|Network Outputs]]<br><br />
}}<br />
Below there is an embedded a neural network classifier, rendered using [https://www.tensorflow.org Tensorflow][http://playground.tensorflow.org Playground]. There are a variety of knobs and buttons on the interface; as we move along, more of these options will become available. Don't worry though, all these will be explained in detail, in due time. For now though, let's define our primary goal throughout this tutorial: '''classification'''.<br />
<br />
The primary task is to train neural nets to classify items into categories, based on some limited information. Like fruit or vegetable; or undergrad major; or Alzheimer's Disease patient (CASE) or control participant (CTRL). In the Tensorflow playground below, you can see a bunch of orange and blue dots. Instead of simply thinking about these as dots at arbitrary spatial coordinates, it will be helpful to think of these as representing people in a clinical study. Let's define the blue dots are patients from the CASE group, and orange dots are CTRL participants. What is our 'limited information' about them? Let's say we have collected information about their age and their score on a dementia screening exam (scores represent number of items forgotten). So the first thing we'd probably want to do is make a scatter plot of these two variables. Let's define their respective dimensions on the plot axes as:<br />
<br />
* x-axis | dim1 | current age ('''''AGE''''')<br />
* y-axis | dim2 | exam score ('''''SCORE''''')<br />
<br />
Notice that after plotting, the dots seem to form clusters. That very promising! If you were asked to draw a line on this plane, to separate these two clusters, it could be easily done. Our brain's neural nets have already solved the the spatial problem. Now let's see if an artificial neural net can solve the same problem. <br />
<br />
Click the blue ''start'' button below; let it run for about 500 epochs (~5 seconds), then click pause.<br />
<br />
{{Clear}}<br />
<br />
===Tensorflow Playground===<br />
<br />
<html><br />
<iframe src="http://playground.tensorflow.org/#activation=tanh&regularization=L1&batchSize=15&dataset=gauss&regDataset=reg-plane&learningRate=0.003&regularizationRate=0.001&noise=0&networkShape=1&seed=0.73576&showTestData=false&discretize=false&percTrainData=40&x=true&y=false&xTimesY=false&xSquared=false&ySquared=false&cosX=false&sinX=false&cosY=false&sinY=false&collectStats=false&problem=classification&initZero=false&hideText=false&showTestData_hide=true&stepButton_hide=true&problem_hide=true&noise_hide=true&discretize_hide=true&regularization_hide=true&dataset_hide=true&batchSize_hide=true&percTrainData_hide=true&regularizationRate_hide=true&learningRate_hide=true&numHiddenLayers_hide=true" height="1050" width="1050" scrolling="no" style="border:none; overflow:hidden; padding-top:-220px; margin-top:-220px;"></iframe> <br />
</html><br />
<br />
{{Clear}}<br />
<br />
Is one neuron with input from ''a single feature'' (the dim1 data: AGE) performing well in the separation task? If so, an orange-colored background should have formed behind the orange dots, while a blue-colored background should have formed behind the blue dots. This colored surface gradient can be understood as the neural network's prediction value at that given coordinate. We will explore prediction values in more detail later on in the tutorial. First let's take a look at what the neural net is taking as inputs.<br />
<br />
{{Clear}}<br />
<br />
===Inputs===<br />
----<br />
{{SmallBox|display=block <br />
|float=right <br />
|clear=none <br />
|width=250px<br />
|margin=5px 2%<br />
|border-width=2px <br />
|border-radius=2px<br />
|[[File: Neural Net Features.png|250px]]<br />
| Figure 1<br />
}}<br />
<br />
To assess the performance, take a look at the input options in ''Figure-1''. There are a bunch of ''X'' variables with subscripts and superscripts, and next to each is a box with various color gradients. For now, let's focus on just two of those symbols, and what they mean to us...<br />
<br />
{| class="wikitable" width=30% align=center <br />
|+ style="font-weight:bold;"|Input Features<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<br />
|}<br />
<br />
These are parsed such that subscripts (''X''<sub>1</sub> , ''X''<sub>2</sub> ,... ''X''<sub>i</sub> ) represent each predictor variable, like AGE and SCORE. As you can see, the first two input options ''X''<sub>1</sub> and ''X''<sub>2</sub> are just ''X''<sub>AGE</sub> and ''X''<sub>SCORE</sub>. Note that since ''X''<sub>1</sub> is plotted on the x-axis, it has a color gradient that changes horizontally, but is constant in the vertical dimension. Conversely the ''X''<sub>2</sub> feature plotted on the y-axis has a vertical color gradient. To clarify why this happens...<br />
<br />
If the only thing we know about these study participants is their AGE, ''X''<sub>1</sub>, we can only make a 1-D plot with each person's age along the x-axis, such that [ x = ''AGE''<sub>i</sub> , y = 0 ]. If you take a look at Figure-2, it should be clear that when information is collapsed onto its single dimension and plotted along the x-axis, the best line we can draw to separate the dim-1 data will be orthogonal to the x-axis (a vertical line). As you move horizontally along the x-axis your categorical guess will likely change, along with the confidence in that guess, which is precisely what is being represented by the color gradient. On the other hand, knowing nothing about exam score, moving up and down on the y-axis will have no effect on your decision, which is why color is constant in the y-dimension. <br />
<br />
When the neural net only gets input about a single feature of each person in the dataset, its synaptic weights will only adapt output along that one dimension. Thus, if for example the network sees that a person is 3 years above the dataset average (considering the data has been ''mean deviated'' and centered), it won't matter what that person's cognitive SCORE was (since the neural net doesn't have access to that info), the network will always make the same guess for anyone 3 years above average age. This is why color is constant at ''x''=3 for any ''y'' value.<br />
<br />
{{Clear}}<br />
<br />
{{SmallBox|display=block <br />
|float=left <br />
|clear=none <br />
|width=420px<br />
|margin=15px 5%<br />
|border-width=2px <br />
|border-radius=2px<br />
|[[File:NN NumberLine.png|400px]]<br />
| Figure 2<br />
}}<br />
<br />
<br />
This isn't a shortcoming of having just one single neuron in the entire network. You could add as many neurons and layers as you want (go ahead and try it)...... if the network only gets input about one feature dimension, the output will be the same, whether there is 1 neuron, or 1 billion. To realize this fact, pretend you can only see the dots as they are plotted in along the number line in 1D (in Figure 2); if we were unable to see the 2D cluster clouds above that line, the billions of neurons in our brain would tell us to draw the classification line in basically the same place as that one single neuron in our artificial neural net. This is a very interesting concept worth noting: neural net classifiers can fail for two very different reasons.<br />
<br />
(1) The neural network itself might be ill-formulated in such a way that, no matter how much information you provide, it cannot seem to learn to solve the classification problem. (2) On the other hand, you might have implemented an apposite deep neural network; yet if the input data is insufficient to solve the classification problem, it will appear to you that this potentially very good neural network performs like garbage. <br><br />
<br />
With that said, there are ways to help prevent that later scenario from happening. These involve doing things like you see for the rest of the input features. The next page will discuss the full set of possible network inputs we have here, which includes...<br />
<br />
<br />
{{Clear}}<br />
<br />
{| class="wikitable" width=30% align=center <br />
|+ style="font-weight:bold;"|Input Features<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub><sup>2</sup> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE<sup>2</sup><br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>2</sub><sup>2</sup> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| SCORE<sup>2</sup><br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| ''X''<sub>1</sub>''X''<sub>2</sub> <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| AGE × SCORE<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| sin(''X''<sub>1</sub>) <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| sin(AGE)<br />
|- style="height:30px"<br />
| style="background:#f7f7f7; border:3px solid #ffffff"| sin(''X''<sub>2</sub>) <br />
|colspan=2 style="background:#f7f7f7; border:3px solid #ffffff"| sin(SCORE)<br />
|}<br />
<br />
<br />
{{Clear}}<br />
{{SmallBox|'''[[Neural Nets 2|Continue to Neural Nets Tutorial Page 2]]'''}}<br />
<br />
<!-- <btn data-toggle="tooltip">Neural Nets 2</btn> --></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Help:Contents&diff=3918Help:Contents2020-05-04T11:35:31Z<p>Monakhos: </p>
<hr />
<div>{{Box|width=255px|MediaWiki [[Help]] Pages|<br />
* [[http://en.wikipedia.org/wiki/Help:Wiki_markup MediaWiki Markup]]<br />
* [[:Category:Templates]]<br />
* [http://www.onesci.com/Help:Contents OneSci Help]<br />
* [http://en.wikipedia.org/wiki/Help:Wiki_markup#Images Mediawiki Markup Images]<br />
* [http://www.mediawiki.org/wiki/Help:Magic_words MediaWiki Magic Words]<br />
* [http://www.mediawiki.org/wiki/Help:Tables MediaWiki Tables]<br />
* [http://commons.wikimedia.org/wiki/Help:Gadget-ImageAnnotator/Installation MediaWiki ImageAnnotator]<br />
* [http://www.mediawiki.org/wiki/Extension:Cite/Cite.php#Separating_references_from_text Mediawiki References]<br />
* [http://www.mediawiki.org/wiki/Manual:Math Math]<br />
* [http://en.wikipedia.org/wiki/Help:Template Template Tips]<br />
* [http://meta.wikimedia.org/wiki/Help:Advanced_templates Advanced Templates]<br />
* [http://en.wikipedia.org/wiki/Wikipedia:Transclusion Transclusion]<br />
}}<br />
<br />
{{Box|width=255px|Bradleymonk.com .JS|<br />
*[[MediaWiki:Common.js]]<br />
*[[MediaWiki:LAPI.js]]<br />
*[[MediaWiki:Gadget-ImageAnnotator.js]]<br />
*[[MediaWiki:ImageAnnotatorConfig.js]]<br />
*[[MediaWiki:LAPI.js]]<br />
*[[MediaWiki:TextCleaner.js]]<br />
*[[MediaWiki:Tooltips.js]]<br />
*[[MediaWiki:UIElements.js]]<br />
*[[MediaWiki:AjaxSubmit.js]]<br />
}}<br />
<br />
{{Box|width=255px|HTML & CSS|<br />
* [https://www.mediawiki.org/wiki/Manual:CSS Manual:CSS]<br />
*: [[MediaWiki:Common.css]]<br />
* [https://www.mediawiki.org/wiki/Manual:Skin_configuration Manual:Skin_configuration]<br />
* [https://www.mediawiki.org/wiki/Manual:Skinning Manual:Skinning]<br />
* [https://www.mediawiki.org/wiki/Help:Skins Help:Skins]<br />
}}<br />
<br />
<br />
{{Clear}}<br />
<br />
<br />
==Lua Scripting==<br />
<br />
* [https://www.mediawiki.org/wiki/Extension:Scribunto Mediawiki Extension:Scribunto]<br />
<br />
* Latest Update: [http://bradleymonk.com/Module:Yesno Modules still not working] < this is a medium-priority TBD when time permits<br />
<br />
== Adding HTML5 Video to MediaWiki ==<br />
I've enabled three options for embedding animated video in this wiki. '''Option 1: PopFig video''' will embed the video a popup box. The video is hidden in a semantic mediawiki icon; mouseover of this icon will pop-open the video. This option makes use of a semantic mediawiki parser tag, secure html extension, and several layers of transclusion. '''Option 2: mediaplayer video''' will embed an inline video using the <code><nowiki><mediaplayer></nowiki></code> tag. The option doesn't require anything but a stand-alone extension. '''Option 3: Widget:Html5media''' makes use of of an Html5 widget. This option is good for very small animations, but does has a fixed size so the portion of a larger videos that extends beyond ~200 pixels will be hidden.<br />
----<br />
<br />
=== Option 1: PopFig ===<br />
<!--<br />
<br> {{PopFig|[[File:Dot.png]]|<html><br />
<video src="http://bradleymonk.com/media2/EXAMPLE.mov" controls></video> <br> <br />
<div style='color:white; width:400px'>This is an example of a movie caption. The text will automatically wrap</div><br />
</html>||<big>SI video1 </big>}}<br />
--><br />
<br />
{{Style|class=div|size=90%|align=left|font=courier|background=#EBFAEB|color=black|pad=8px|margin=6px|<br />
<nowiki> <br />
{{PopFig|[[File:Dot.png]]|<html><br />
<br />
<video src="http://bradleymonk.com/media2/EXAMPLE.mov" controls></video> <br> <br />
<br />
<div style='color:white; width:400px'>This is an example of a movie caption. The text will automatically wrap</div><br />
<br />
</html>||<big>SI video1 </big>}}<br />
</nowiki> <br />
}}<br />
----<br />
<br />
=== Option 2: mediaplayer ===<br />
<br> <mediaplayer image='http://bradleymonk.com/media2/EXAMPLE.png' width='500' height='300'>http://bradleymonk.com/media2/EXAMPLE.mov</mediaplayer><br />
<br />
{{Style|class=div|size=90%|align=left|font=courier|background=#EBFAEB|color=black|pad=8px|margin=6px|<br />
<nowiki> <br />
<mediaplayer image='http://bradleymonk.com/media2/EXAMPLE.png' width='500' height='300'>http://bradleymonk.com/media2/EXAMPLE.mov</mediaplayer> <br />
</nowiki><br />
}}<br />
<br />
If the video is uploaded using the native wiki upload link in the sidebar, this option can be employed simply by pasting the [[Example uploaded video|uploaded video]] file name between the <code><nowiki><mediaplayer> </mediaplayer></nowiki></code> tags like so:<br />
<br />
{{Style|class=div|size=90%|align=left|font=courier|background=#EBFAEB|color=black|pad=8px|margin=6px|<br />
<nowiki> <br />
<mediaplayer image='http://www.bradleymonk.com/w/images/8/82/RenderTorus.jpg' width='500' height='300'>File:RenderTorus.mp4</mediaplayer><br />
</nowiki><br />
}}<br />
<br />
<br />
<mediaplayer image='http://www.bradleymonk.com/w/images/8/82/RenderTorus.jpg' width='500' height='300'>File:RenderTorus.mp4</mediaplayer><br />
----<br />
<br />
=== Option 3: [[Widget:Html5media|Widget:Html5media]] ===<br />
<br> {{Box|font=90%|width=45%|float=left|text=12px|boarder=solid #aaa 1px|Embeded Version|<br />
<br />
{{#widget:Html5media<br />
|url=http://bradleymonk.com/media2/DIFFUSE.mp4<br />
|width=200<br />
|height=200<br />
}}<br />
<br />
<pre><br />
{{#widget:Html5media<br />
|url=http://bradleymonk.com/media2/DIFFUSE.mp4<br />
|width=200<br />
|height=200<br />
}}<br />
</pre><br />
<br />
}}<!-- END BOX --><br />
<br />
{{Box|font=120%|width=45%|float=left|text=12px|boarder=solid #aaa 1px|Popup Version|<br />
<br />
Hover mouse over icon to open: {{Popup|{{#widget:Html5media|url=http://bradleymonk.com/media2/DIFFUSE.mp4|width=200|height=200}}}}<br />
<br />
<br />
<pre><br />
{{Popup|{{#widget:Html5media<br />
|url=http://bradleymonk.com/media2/DIFFUSE.mp4<br />
|width=200<br />
|height=200<br />
}}}}<br />
</pre><br />
<br />
}}<!-- END BOX --><br />
<br />
{{Clear}}<br />
<br />
==Semantic Mediawiki Popups==<br />
<br />
<big>[http://semantic-mediawiki.org/wiki/Help:Adding_tooltips Semantic Mediawiki Tool Tips]</big><br />
<br />
The parser function '''#info''' allows to add tooltips containing additional information or warnings.<br />
<br />
<big>Syntax</big><br><br />
<br />
<br />
* <code><nowiki>{{#info: <text> | <icon> }}</nowiki></code><br />
<br />
* you must include the "nowiki" tag around any equal signs if you want to use styles inside the into popup <br />
** example: <code><nowiki><span style</nowiki><nowiki><nowiki>=</nowiki></nowiki><nowiki>"color:red"></nowiki></code> <br />
** result {{#info:text <span style<nowiki>=</nowiki>"color:red">this is red</span> text|warning}} <br />
* <code><nowiki>{{#info:text <span style<nowiki>=</nowiki>"color:red">this is red</span> text}}</nowiki></code><br />
<br />
<br />
* If you want to have a large image popup, the best way to do this is using these inline styles with the span tag<br />
** <code>position:fixed; left:10px; top:10px; overflow:visible;</code><br />
** inline text {{#info:text <span style<nowiki>=</nowiki>"position:fixed; left:10px; top:10px; overflow:visible;">THIS WILL PUT THE IMAGE UP HERE</span> text}} inline text<br />
** Use the Popupimg template inside the Popup template to do exactly this<br />
<br />
<!--<br />
** example: <code><nowiki>{{#info: {{Popupimg|[[File:Nicoll2011 Fig1.png]]}} }}</nowiki></code><br />
** result: {{#info: {{Popupimg|[[File:Nicoll2011 Fig1.png]]}} }}<br />
** example: <code><nowiki>{{Popup| {{Popupimg|[[File:Nicoll2011 Fig1.png]] }} }}</nowiki></code><br />
** result: {{Popup| {{Popupimg|[[File:Nicoll2011 Fig1.png]]}} }}<br />
--><br />
<br />
==Extension LinkTitles==<br />
<br />
;AUTO GENERATED SITE LINKS<br />
<br />
* [http://www.mediawiki.org/wiki/Extension:LinkTitles MediaWiki Extension Page]<br />
<br />
<pre><br />
require_once( "$IP/extensions/LinkTitles/LinkTitles.php" );<br />
$wgLinkTitlesSmartMode = false; // false = case sensitive<br />
$wgLinkTitlesMinimumTitleLength = 7; // default is 3<br />
$wgLinkTitlesSkipTemplates = true; // skip any text in a {{template|text}}<br />
$wgLinkTitlesFirstOnly = true; // only link the first occurrence<br />
$wgLinkTitlesBlackList = array();<br />
$wgLinkTitlesBlackList[] = 'MediaWiki';<br />
$wgLinkTitlesBlackList[] = 'mediaplayer';<br />
</pre><br />
<br />
; USE THE __NOAUTOLINKS__ TAG ON A WIKI PAGE TO PREVENT LINKING ON THAT PAGE<br />
<br />
<pre><br />
__NOAUTOLINKS__ <br />
</pre><br />
<br />
__NOAUTOLINKS__<br />
<br />
==Template:SlideBox==<br />
<nowiki><br />
{{SlideBox|width=80%|float=left|<br />
Header [[Content]] Is Shown<br />
|<br />
Body text is hidden<br />
}}<br />
</nowiki><br />
<br />
==Customize Editing Toolbar==<br />
http://bradleymonk.com/User:Bradley_Monk/common.js<br />
http://www.mediawiki.org/wiki/Extension:WikiEditor/Toolbar_customization<br />
<br />
;section<br />
*{String} (optional) The name of the section in the WikiEditor. Defaults to 'main'<br />
;id<br />
*{String} (required) Unique id (ie. 'my-button')<br />
;icon<br />
*{String} (recommended) URL to the icon, should be square about 21 to 22px<br />
;label<br />
*{String} (required) Tooltip displayed when hovering button<br />
;insertBefore<br />
*{String} (optional) Wikitext to be inserted before the cursor on-click<br />
;sampleText<br />
*{String} (optional) Text inserted in place of the cursor if no text was selected<br />
;insertAfter<br />
*{String} (optional) Wikitext to be inserted after the cursor on-click<br />
;callback<br />
*{Function} (optional) Called when the button is clicked<br />
;autoSummary<br />
*{mixed} (optional) Null or an Object with the following properties:<br />
**summary: {String} (required) Edit summary that should be used<br />
**position: {String} (optional) 'append', 'prepend' or 'replace'<br />
**delimiter: {String} (optional) delimiter between the (possibly) current summary and the to-be-inserted summary<br />
<br />
==Syntax Highlighting==<br />
The '''Extension:SyntaxHighlight GeSHi''' tag displays formatted [[w:source code|source code]] with the <code>&lt;syntaxhighlight&gt;</code> tag.<br />
<br />
This extension also adds coloring according to the code language settings. Like the <code>&lt;pre&gt;</code> tags and the <code>&lt;[[Extension:Poem|poem]]&gt;</code> tags, the tags shows the coding exactly as it was typed, preserving white space. <br />
<br />
This extension also can create line numbers.<br />
<br />
=== Usage ===<br />
On the wiki page, you can now use "syntaxhighlight" elements:<br />
<br />
<pre><nowiki><br />
<syntaxhighlight lang="php"><br />
<?php<br />
$v = "string"; // sample initialization<br />
?><br />
html text<br />
<?<br />
echo $v; // end of php code<br />
?><br />
</syntaxhighlight><br />
</nowiki></pre><br />
<br />
shows:<br />
<syntaxhighlight lang="php"><br />
<?php<br />
$v = "string"; // sample initialization<br />
?><br />
html text<br />
<?<br />
echo $v; // end of php code<br />
?><br />
</syntaxhighlight><br />
<br />
=== Alternative &lt;source&gt; tag ===<br />
Before [[rev:50696]], the extension used the tag <code>[[Extension:SyntaxHighlighter|&lt;source&gt;]]</code>.<br />
This is still supported, but <code>&lt;syntaxhighlight&gt;</code> avoids conflicts if your source code itself contains <nowiki><source></nowiki> (for example XML).<br />
<br />
=== Parameters ===<br />
; <code>lang="??"</code><br />
: Defines what programming language the source code is using. This affects how the extension highlights the source code. See the section "[[#Supported_languages|Supported languages]]" in this page for details of supported languages.<br />
; <code>line="GESHI_NORMAL_LINE_NUMBERS|GESHI_FANCY_LINE_NUMBERS"</code><br />
: Type of line numbering to use. If you do not provide this parameter, then lines will not be numbered. Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#enabling-line-numbers enable_line_numbers] the flag in GeSHi.<br />
; <code>line</code><br />
: Equivalent to <code>'''line="GESHI_FANCY_LINE_NUMBERS"</code><br />
; <code>line start="??"</code><br />
: Use together with the parameter "'''line'''". Define the start number of the line. If you type line start="55", it will start counting at 55, then 56,57,58... and so on. Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#starting-line-numbers start_line_numbers_at] method on GeSHi<br />
<!--<br />
; <code>case</code><br />
: Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#auto-caps-nocaps set_case_keywords] method on GeSHi<br />
; <code>tab</code><br />
: Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#setting-tab-width set_tab_width] method on GeSHi<br />
--><br />
; <code>highlight="??"</code><br />
: Specifies which line is highlighted. Note that the parameter '''line start="??"''' doesn't affect how it counts the lines. <br />
; <code>enclose="??"</code><br />
: Specifies what container is used to enclose the source code. Takes values "pre" (default value), "div", "none". Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#the-code-container set_header_type] method on GeSHi. Choosing "div" will cause text to wrap, which is helpful if text is extending off the edge of the screen, causing horizontal scrolling.<br />
; <code>strict</code><br />
: Type the name of the parameter to enable the strict mode. Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#using-strict-mode enable_strict_mode] method on GeSHi.<br />
<br />
The effect and usage of these parameters can be consulted in [http://qbnz.com/highlighter/geshi-doc.html GeSHi's documentation].<br />
<br />
Since r22246, you can override the colors using '''MediaWiki:Geshi.css'''.<br />
<br />
=== More Usage ===<br />
When line numbering is added with ''line'', long code lines will be wrapped. See the example below. When ''text'' is the selected language, and numbering is used, the behaviour resembles the use of ''pre'' tags with numbering and long-line wrapping.<br />
<br />
The following example shows how to color an HTML code listing:<br />
<pre><syntaxhighlight lang="html4strict" line start="100" highlight="5" enclose="div"><br />
HTML module goes here...<br />
</syntaxhighlight><br />
</pre> <br />
A typical result is just: <br />
<syntaxhighlight lang="html4strict" line start="100" highlight="5" enclose="div"><br />
<!--This is a comment. Comments are not displayed in the browser--><br />
<table align=center style="background: ivory;color:maroon;font-style:italic;font-family:arial;font-weight:bold;font-size:10pt;"> <br />
<tr><th> Heading 1 </th><th> Heading 2 </th></tr><br />
<tr><br />
<td style="padding:10px;"> This is cell 1 text </td><br />
<td style="padding:10px;"> This is cell 2 text </td><br />
</tr><br />
</table><br />
</syntaxhighlight><br />
<br />
=== Supported languages ===<br />
These are the languages known by GeSHi that can be used in the '''lang''' parameter.<br />
<br />
<small>Note that installed GeSHi version (can be revealed by previewing e.g. <code>&lt;syntaxhighlight lang="-"&gt;&lt;/syntaxhighlight&gt;</code>) might not be the most recent version; see [[bugzilla:10967]] for Wikimedia sites.</small><br />
<br />
<small>The GeSHi sources for each language can be found in [http://sourceforge.net/p/geshi/code/HEAD/tree/trunk/geshi-1.0.X/src/geshi/ VCS on SourceForge]. Remember to go and select the version that the MediaWiki installation uses.</small><br />
<br />
{| class="wikitable" style="float:left"<br />
!Code||Language<br />
|-<br />
|<code>abap</code>||[[:en:ABAP|ABAP]]<br />
|-<br />
|<code>actionscript</code>||[[:en:ActionScript|ActionScript]]<br />
|-<br />
|<code>ada</code>||[[:en:Ada (programming language)|Ada]]<br />
|-<br />
|<code>apache</code>||[[:en:Apache HTTP Server|Apache Configuration]]<br />
|-<br />
|<code>applescript</code>||[[:en:AppleScript|AppleScript]]<br />
|-<br />
|<code>asm</code>||[[:en:Assembly language|Assembly]]<br />
|-<br />
|<code>asp</code>||[[:en:Active Server Pages|Active Server Pages (ASP)]]<br />
|-<br />
|<code>autoit</code>||[[:en:AutoIt|AutoIt]]<br />
|-<br />
|<code>bash</code>||[[:en:Bash (Unix shell)|Bash]]<br />
|-<br />
|<code>basic4gl</code>||[[:en:Basic4GL|Basic4GL]]<br />
|-<br />
|<code>bf</code>||[[:en:Brainfuck|Brainfuck]]<br />
|-<br />
|<code>blitzbasic</code>||[[:en:Blitz BASIC|Blitz BASIC]]<br />
|-<br />
|<code>bnf</code>||[[:en:Backus-Naur Form|Backus-Naur Form]]<br />
|-<br />
|<code>c</code>||[[:en:C (programming language)|C]]<br />
|-<br />
|<code>c_mac</code>||C (Mac)<br />
|-<br />
|<code>caddcl</code>||[[:en:Dialog Control Language|AutoCAD DCL]]<br />
|-<br />
|<code>cadlisp</code>||[[:en:AutoLISP|AutoLISP]]<br />
|-<br />
|<code>cfdg</code>||CFDG<br />
|-<br />
|<code>cfm</code>||[[:en:ColdFusion_Markup_Language|ColdFusion Markup Language]]<br />
|-<br />
|<code>cil</code>||[[:en:Common_Intermediate_Language|Common Intermediate Language (CIL)]]<br />
|-<br />
|<code>cobol</code>||[[:en:COBOL|COBOL]]<br />
|-<br />
|<code>cpp-qt</code>||[[:en:Qt (toolkit)|C++ (Qt toolkit)]]<br />
|-<br />
|<code>cpp</code>||[[:en:C++|C++]]<br />
|-<br />
|<code>csharp</code>||[[:en:C Sharp (programming language)|C#]]<br />
|-<br />
|<code>css</code>||[[:en:Cascading Style Sheets|Cascading Style Sheets (CSS)]]<br />
|-<br />
|<code>d</code>||[[:en:D (programming language)|D]]<br />
|-<br />
|<code>delphi</code>||[[:en:Delphi programming language|Delphi]]<br />
|-<br />
|<code>diff</code>||[[:en:diff|Diff]]<br />
|-<br />
|<code>div</code>||DIV<br />
|-<br />
|<code>dos</code>||[[:en:DOS batch file|DOS batch file]]<br />
|-<br />
|<code>dot</code>||[[:en:DOT language|DOT]]<br />
|-<br />
|<code>eiffel</code>||[[:en:Eiffel (programming language)|Eiffel]]<br />
|-<br />
|<code>fortran</code>||[[:en:Fortran|Fortran]]<br />
|-<br />
|<code>freebasic</code>||[[:en:FreeBASIC|FreeBASIC]]<br />
|-<br />
|<code>gambas</code>||[[:en:Gambas_programming_language|Gambas]]<br />
|-<br />
|<code>genero</code>||Genero<br />
|-<br />
|<code>gettext</code>||[[:en:GNU_gettext|GNU internationalization (i18n) library]]<br />
|-<br />
|<code>glsl</code>||[[:en:GLSL|OpenGL Shading Language (GLSL)]]<br />
|-<br />
|<code>gml</code>||[[:en:Game Maker Language|Game Maker Language (GML)]]<br />
|-<br />
|<code>gnuplot</code>||[[:en:Gnuplot|gnuplot]]<br />
|-<br />
|<code>groovy</code>||[[:en:Groovy (programming language)|Groovy]]<br />
|-<br />
|<code>haskell</code>||[[:en:Haskell (programming language)|Haskell]]<br />
|-<br />
|<code>hq9plus</code>||HQ9+<br />
|-<br />
|<code>html4strict</code>||[[:en:HTML|HTML]]<br />
|-<br />
|<code>html5</code>||[[:en:HTML5|HTML5]]<br />
|-<br />
|<code>idl</code>||[[:en:Universal Network Objects|Uno IDL]]<br />
|-<br />
|<code>ini</code>||[[:en:INI file|INI]]<br />
|-<br />
|<code>inno</code>||[[:en:Inno Setup|Inno]]<br />
|-<br />
|<code>intercal</code>||[[:en:INTERCAL|INTERCAL]]<br />
|-<br />
|<code>io</code>||[[:en:Io (programming language)|Io]]<br />
|-<br />
|<code>java</code>||[[:en:Java (programming language)|Java]]<br />
|-<br />
|<code>java5</code>||[[:en:Java (programming language)|Java(TM) 2 Platform Standard Edition 5.0 ]]<br />
|-<br />
|<code>javascript</code>||[[:en:JavaScript|JavaScript]]<br />
|-<br />
|<code>kixtart</code>||[[:en:KiXtart|KiXtart]]<br />
|-<br />
|<code>klonec</code>||Klone C<br />
|-<br />
|<code>klonecpp</code>||Klone C++<br />
|-<br />
|<code>latex</code>||[[:en:LaTeX|LaTeX]]<br />
|-<br />
|<code>lisp</code>||[[:en:Lisp (programming language)|Lisp]]<br />
|-<br />
|<code>lolcode</code>||[[:en:LOLCODE|LOLCODE]]<br />
|-<br />
|<code>lotusscript</code>||[[:en:LotusScript|LotusScript]]<br />
|-<br />
|<code>lua</code>||[[:en:Lua (programming language)|Lua]]<br />
|}<br />
{| class="wikitable" style="float:left"<br />
!Code||Language<br />
|-<br />
|<code>m68k</code>||[[:en:Motorola 68000|Motorola 68000 Assembler]]<br />
|-<br />
|<code>make</code>||[[:en:Make_(software)|make]]<br />
|-<br />
|<code>matlab</code>||[[:en:MATLAB|MATLAB M]]<br />
|-<br />
|<code>mirc</code>||[[:en:mIRC scripting language|mIRC scripting language]]<br />
|-<br />
|<code>mxml</code>||[[:en:MXML|MXML]]<br />
|-<br />
|<code>mpasm</code>||[[:en:PIC microcontroller|Microchip Assembler]]<br />
|-<br />
|<code>mysql</code>||[[:en:MySQL|MySQL]]<br />
|-<br />
|<code>nsis</code>||[[:en:Nullsoft Scriptable Install System|Nullsoft Scriptable Install System (NSIS)]]<br />
|-<br />
|<code>objc</code>||[[:en:Objective-C|Objective-C]]<br />
|-<br />
|<code>ocaml-brief</code>||[[:en:Objective Caml|OCaml]]<br />
|-<br />
|<code>ocaml</code>||[[:en:Objective Caml|OCaml]]<br />
|-<br />
|<code>oobas</code>||[[:en:StarOffice Basic|OpenOffice.org Basic]]<br />
|-<br />
|<code>oracle8</code>||[[:en:PL/SQL|Oracle 8 SQL]]<br />
|-<br />
|<code>oracle11</code>||[[:en:PL/SQL|Oracle 11 SQL]]<br />
|-<br />
|<code>pascal</code>||[[:en:Pascal (programming language)|Pascal]]<br />
|-<br />
|<code>per</code>||per<br />
|-<br />
|<code>perl</code>||[[:en:Perl|Perl]]<br />
|-<br />
|<code>php-brief</code>||[[:en:PHP|PHP]]<br />
|-<br />
|<code>php</code>||[[:en:PHP|PHP]]<br />
|-<br />
|<code>pixelbender</code>||[[:en:Adobe_Pixel_Bender|Pixel Bender]]<br />
|-<br />
|<code>plsql</code>||[[:en:PL/SQL|PL/SQL]]<br />
|-<br />
|<code>povray</code>||[[:en:POV-Ray|Persistence of Vision Raytracer]]<br />
|-<br />
|<code>powershell</code>||[[:en:Windows_PowerShell|Windows PowerShell]]<br />
|-<br />
|<code>progress</code>||[[:en:OpenEdge_Advanced_Business_Language|OpenEdge Advanced Business Language]]<br />
|-<br />
|<code>prolog</code>||[[:en:Prolog|Prolog]]<br />
|-<br />
|<code>providex</code>||[[:en:ProvideX|ProvideX]]<br />
|-<br />
|<code>python</code>||[[:en:Python (programming language)|Python]]<br />
|-<br />
|<code>qbasic</code>||[[:en:QBasic|QBasic/QuickBASIC]]<br />
|-<br />
|<code>rails</code>||[[:en:Ruby on Rails|Rails]]<br />
|-<br />
|<code>reg</code>||[[:en:Windows Registry|Windows Registry]]<br />
|-<br />
|<code>robots</code>||[[:en:Robots Exclusion Standard|robots.txt]]<br />
|-<br />
|<code>rsplus</code>||[[:en:R (programming language)|R]]<br />
|-<br />
|<code>ruby</code>||[[:en:Ruby (programming language)|Ruby]]<br />
|-<br />
|<code>sas</code>||[[:en:SAS System|SAS]]<br />
|-<br />
|<code>scala</code>||[[:en:Scala_(programming_language)|Scala]]<br />
|-<br />
|<code>scheme</code>||[[:en:Scheme (programming language)|Scheme]]<br />
|-<br />
|<code>scilab</code>||[[:en:Scilab|Scilab]]<br />
|-<br />
|<code>sdlbasic</code>||[[:en:SdlBasic|SdlBasic]]<br />
|-<br />
|<code>smalltalk</code>||[[:en:Smalltalk|Smalltalk]]<br />
|-<br />
|<code>smarty</code>||[[:en:Smarty|Smarty]]<br />
|-<br />
|<code>sql</code>||[[:en:SQL|SQL]]<br />
|-<br />
|<code>tcl</code>||[[:en:Tcl|Tcl]]<br />
|-<br />
|<code>teraterm</code>||[[:en:TeraTerm|Tera Term]]<br />
|-<br />
|<code>text</code>||[[:en:Plain text|Plain text]]<br />
|-<br />
|<code>thinbasic</code>||[[:en:thinBasic|thinBasic]]<br />
|-<br />
|<code>tsql</code>||[[:en:Transact-SQL|Transact-SQL]]<br />
|-<br />
|<code>typoscript</code>||[[:en:TYPO3|TypoScript]]<br />
|-<br />
|<code>vala</code>||[[:en:Vala_(programming_language)|Vala]]<br />
|-<br />
|<code>vb</code>||[[:en:Visual Basic|Visual Basic]]<br />
|-<br />
|<code>vbnet</code>||[[:en:Visual Basic .NET|Visual Basic .NET]]<br />
|-<br />
|<code>verilog</code>||[[:en:Verilog|Verilog]]<br />
|-<br />
|<code>vhdl</code>||[[:en:VHSIC Hardware Description Language|VHDL]]<br />
|-<br />
|<code>vim</code>||[[:en:Vimscript|Vimscript]]<br />
|-<br />
|<code>visualfoxpro</code>||[[:en:Visual FoxPro|Visual FoxPro]]<br />
|-<br />
|<code>visualprolog</code>||[[:en:Visual_Prolog|Visual Prolog]]<br />
|-<br />
|<code>whitespace</code>||[[:en:Whitespace_(programming_language)|Whitespace]]<br />
|-<br />
|<code>winbatch</code>||[[:en:Winbatch|Winbatch]]<br />
|-<br />
|<code>xml</code>||[[:en:XML|XML]]<br />
|-<br />
|<code>xorg_conf</code>||[[:en:Xorg.conf|Xorg.conf]]<br />
|-<br />
|<code>xpp</code>||[[:en:Microsoft_Dynamics_AX|X++]]<br />
|-<br />
|<code>z80</code>||[[:en:Zilog Z80|ZiLOG Z80 Assembler]]<br />
|}<br />
<div style="clear:both"></div><br />
<br />
=== Extra features ===<br />
<br />
; Default Source Language<br />
<br />
Added in [[rev:50693]].<br />
<br />
If the site mainly quotes the source code of a specific programming language, it would be helpful to set a default language.<br />
<br />
To do so, add a new variable to LocalSettings.php, just after the require_once line. We set [[:en:C (programming language)|C programming language]] as an example:<br />
<syntaxhighlight lang="php"><br />
require_once("$IP/extensions/SyntaxHighlight_GeSHi/SyntaxHighlight_GeSHi.php");<br />
$wgSyntaxHighlightDefaultLang = "c";<br />
</syntaxhighlight><br />
<br />
==LaTeX Math==<br />
<br />
; NEW WAY<br />
<br />
<pre><tex>f{(x)} = {\sum \lambda \cdot \bar{y} \over \sum \lambda^{ \over 2 n} }</tex></pre><br />
<br />
:: <tex>f{(x)} = {\sum \lambda \cdot \bar{y} \over \sum \lambda^{ \over 2 n} }</tex><br />
<br />
; OLD WAY<br />
<br />
<pre><br />
<math> f(x, \mu, \sigma) = \frac{1}{\sigma \sqrt{2\pi} } e^{ -\frac{(x-\mu)^2}{2\sigma^2} } </math><br />
</pre><br />
<br />
::<math><br />
f(x, \mu, \sigma) = \frac{1}{\sigma \sqrt{2\pi} } e^{ -\frac{(x-\mu)^2}{2\sigma^2} }<br />
</math><br />
<br />
; {{TeX}} and HTML<br />
<br />
<big>[http://en.wikipedia.org/wiki/Table_of_mathematical_symbols LIST OF MATH SYMBOLS IN HTML MARKUP]</big><br><br />
<br />
{| class="wikitable"<br />
|-<br />
! {{TeX}} Syntax ([[#Forced_PNG_rendering|forcing PNG]])<br />
! {{TeX}} Rendering<br />
! HTML Syntax<br />
! HTML Rendering<br />
|-<br />
| <code><nowiki><math>\alpha</math></nowiki></code><br />
| <math>\alpha</math><br />
| <code><nowiki>{{math|<VAR>&amp;alpha;</VAR>}}</nowiki></code><br />
| {{math|<VAR>&alpha;</VAR>}}<br />
|-<br />
| <code><nowiki><math> f(x) = x^2\,</math></nowiki></code><br />
| <math> f(x) = x^2\,</math><br />
| <code><nowiki>{{math|''f''(<var>x</var>) {{=}} <var>x</var><sup>2</sup>}}</nowiki></code><br />
| {{math|''f''(<var>x</var>) {{=}} <var>x</var><sup>2</sup>}}<br />
|-<br />
| <code><nowiki><math>\sqrt{2}</math></nowiki></code><br />
| <math>\sqrt{2}</math><br />
| <code><nowiki>{{math|{{radical|2}}}}</nowiki></code><br />
| {{math|{{radical|2}}}}<br />
|-<br />
| <code><nowiki><math>\sqrt{1-e^2}</math></nowiki></code><br />
| <math>\sqrt{1-e^2}</math><br />
| <code><nowiki>{{math|{{radical|1 &amp;minus; ''e''&amp;sup2;}}}}</nowiki></code><br />
| {{math|{{radical|1 &minus; ''e''&sup2;}}}}<br />
|}<br />
<br />
The codes on the left produce the symbols on the right, but the latter can also be put directly in the wikitext, except for &lsquo;=&rsquo;.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Syntax<br />
! Rendering<br />
|- valign="top"<br />
|<pre><nowiki>&amp;alpha; &amp;beta; &amp;gamma; &amp;delta; &amp;epsilon; &amp;zeta;<br />
&amp;eta; &amp;theta; &amp;iota; &amp;kappa; &amp;lambda; &amp;mu; &amp;nu;<br />
&amp;xi; &amp;omicron; &amp;pi; &amp;rho; &amp;sigma; &amp;sigmaf;<br />
&amp;tau; &amp;upsilon; &amp;phi; &amp;chi; &amp;psi; &amp;omega;<br />
&amp;Gamma; &amp;Delta; &amp;Theta; &amp;Lambda; &amp;Xi; &amp;Pi;<br />
&amp;Sigma; &amp;Phi; &amp;Psi; &amp;Omega;<br />
</nowiki></pre><br />
| style="texhtml" |α β γ δ ε ζ<br <br />
/>η θ ι κ λ μ ν<br <br />
/>ξ ο π ρ σ ς<br <br />
/>τ υ φ χ ψ ω<br<br />
/>Γ Δ Θ Λ Ξ Π<br<br />
/>Σ Φ Ψ Ω<br />
|- valign="top"<br />
| valign="middle" | <pre><nowiki>&amp;int; &amp;sum; &amp;prod; &amp;radic; &amp;minus; &amp;plusmn; &amp;infty;<br />
&amp;asymp; &amp;prop; {{=}} &amp;equiv; &amp;ne; &amp;le; &amp;ge; <br />
&amp;times; &amp;middot; &amp;divide; &amp;part; &amp;prime; &amp;Prime;<br />
&amp;nabla; &amp;permil; &amp;deg; &amp;there4; &amp;Oslash; &amp;oslash;<br />
&amp;isin; &amp;notin; <br />
&amp;cap; &amp;cup; &amp;sub; &amp;sup; &amp;sube; &amp;supe;<br />
&amp;not; &amp;and; &amp;or; &amp;exist; &amp;forall; <br />
&amp;rArr; &amp;hArr; &amp;rarr; &amp;harr; &amp;uarr; <br />
&amp;alefsym; - &amp;ndash; &amp;mdash; <br />
</nowiki></pre><br />
| style="texhtml" |∫ ∑ ∏ √ − ± ∞<br <br />
/>≈ ∝ = ≡ ≠ ≤ ≥<br <br />
/>× · ÷ ∂ ′ ″<br <br />
/>∇ ‰ ° ∴ Ø ø<br<br />
/>∈ ∉ ∩ ∪ ⊂ ⊃ ⊆ ⊇<br<br />
/>¬ ∧ ∨ ∃ ∀<br <br />
/>⇒ ⇔ → ↔ ↑<br <br />
/>ℵ - – —<br />
|}<br />
<br />
==Image Annoation==<br />
<br />
<nowiki><br />
<div class="wpImageAnnotatorEnable"><br />
<span class="wpImageAnnotatorPageName" style="display:none;">[[Sandbox]]</span><br />
<span class="wpImageAnnotatorFullName" style="display:none;">[[Sandbox]]</span><br />
<div class="wpImageAnnotatorFile">[[File:Foo.png]]</div><br />
<div style="display:none;"><div><div><!--IMAGE NOTES BELOW -->{{ImageNoteEnd|id=-1}}<br />
</div><br />
</nowiki><br />
<br />
==Livelets==<br />
*[http://www.mediawiki.org/wiki/Extension:Livelets Livelets]<br />
<br />
To create livelets, wrap any areas of the page in the #live parser-function.<br />
<br />
In the following example, a transclusion of the page called Template:MyVerySlowTable has been turned into a livelet:<br />
<br />
<nowiki><br />
{{#live: {{MyVerySlowTable}} }}<br />
</nowiki><br />
<br />
<br />
==References and Citations==<br />
<br />
===Using Native Ref Tags===<br />
In-text references make it easy to copy the text to another page; on the other hand, they make it hard to read. References containing a lot of data, quotes or elaborate citation templates can make up a significantly larger fraction of the source than the text that will actually be visible. To avoid this, recent versions of the extension allow moving some or all of the references into the <code>&lt;references /></code> section, to the place where they will actually appear to the reader. Thus,<br />
<br />
<blockquote><br />
<tt><br />
According to scientists, the Sun is pretty big.<font color=red>'''&lt;ref name="miller" />'''</font><br /><br />
The Moon, however, is not so big.<font color=red>'''&lt;ref name="smith" />'''</font><br /><br />
<br /><br />
&#61;=Notes==<br />
<br /><br />
<font color=red>'''&lt;references>'''<br /><br />
'''&lt;ref name="miller">'''E. Miller, ''The Sun'', (New York: Academic Press, 2005), 23-5.'''&lt;/ref>'''</br><br />
'''&lt;ref name="smith">'''R. Smith, "Size of the Moon", ''Scientific American'', 46 (April 1978): 44-6.'''&lt;/ref>'''<br /><br />
'''&lt;/references>'''</font><br />
</tt><br />
</blockquote><br />
<br />
will have the same output as the [[#Example|first example]] above, although the numbering and order of the references will not in general be the same.<br />
<br />
----<br />
==HTML ==<br />
<br />
===Example===<br />
<br />
<html><br />
<iframe src="http://www.w3schools.com/tags/tag_iframe.asp" height="100" width="400" frameborder="1" seamless="seamless" style="float:left"></iframe><br />
</html><br />
<br />
{{Clear}}<br />
{{Box|width=600px|iFrame Code|<br />
<code><br />
<nowiki><br />
<html><br />
<iframe src="http://www.w3schools.com/tags/tag_iframe.asp" <br />
height="300" width="350" frameborder="1" seamless="seamless" style="float:left"><br />
</iframe><br />
</html><br />
</nowiki><br />
</code><br />
}}<br />
<br />
{{Clear}}<br />
===iFrame Attributes===<br />
<br />
{| class="wikitable"<br />
|-<br />
! Attribute !! Value !! Description<br />
|-<br />
| align || left, right, top, middle, bottom || Specifies the alignment of an iframe according to surrounding elements<br />
|-<br />
| frameborder || 1, 0 || Specifies whether or not to display a border around an iframe<br />
|-<br />
| height || pixels || Specifies the height of an iframe<br />
|-<br />
| longdesc || URL || Specifies a page that contains a long description of the [[content]] of an iframe<br />
|-<br />
| marginheight || pixels || Specifies the top and bottom margins of the [[content]] of an iframe<br />
|-<br />
| marginwidth || pixels || Specifies the left and right margins of the [[content]] of an iframe<br />
|-<br />
| name || name || Specifies the name of an iframe<br />
|-<br />
| [[sandbox]] || "", allow-forms, allow-same-origin, allow-scripts, allow-top-navigation || Enables a set of extra restrictions for the [[content]] in the iframe<br />
|-<br />
| scrolling || yes, no, auto || Specifies whether or not to display scrollbars in an iframe<br />
|-<br />
| seamless || seamless || Specifies that the iframe should look like it is a part of the containing document<br />
|-<br />
| src || URL || Specifies the address of the document to embed in the iframe<br />
|-<br />
| srcdoc || HTML_code || Specifies the HTML [[content]] of the page to show in the iframe<br />
|-<br />
| width || pixels || Specifies the width of an iframe<br />
|}<br />
<br />
==Tables==<br />
<br />
===wikitable===<br />
{| class="wikitable" style="background-color:white;"<br />
|-<br />
! Link !! Description<br />
|-<br />
|[http://vcell.org/bionetgen/ BioNetGen] || A web-based tool for automatically generating a biochemical reaction networks from user-specified rules for biomolecular interactions. <br />
|-<br />
|[http://bionumbers.hms.harvard.edu/ B10NUMB3R5] || (BIONUMBERS) A database of useful biological numbers. <br />
|-<br />
|[http://www.bioinformaticsservices.com/ BioInformatics Services] || BioInformatics Services is a biotechnology consulting firm specializing in using computational biology to solve complex biological and data analysis problems. <br />
|-<br />
|[http://www.cellmigration.org/resource/modeling/model_resources.shtml#softwares cellmigrationgateway] || modeling software <br />
|-<br />
|[http://www.cellml.org/ CellML] || The CellML language is an open standard based on the XML markup language at the University of Auckland and affiliated research groups. <br />
|-<br />
|[https://sites.google.com/site/compcellbio/ CompCell Bio Web] || Development site for teaching modules on quantitative cell biology. <br />
|-<br />
|[http://doqcs.ncbs.res.in/ DOQCS] || Quantitative Cellular Signaling (DOQCS) is a repository of models of signaling pathways. <br />
|-<br />
|[http://www.e-cell.org/ecell/ E-Cell] || A Multi-Algorithm, Multi-Timescale Simulation Software Environment. <br />
|-<br />
|[http://gfit.sourceforge.net/ Gfit] || A program for global regression analysis of experimental data. <br />
|-<br />
|[http://www.kitware.com/ Kitware] || Professional Visualization Solutions, Tools and Support. <br />
|-<br />
|[http://www.mcell.cnl.salk.edu/ MCell] || General Monte Carlo Simulator of Cellular Microphysiology. <br />
|-<br />
|[http://mnm.engr.uconn.edu/MNM/SMSSearchServlet Minimotif Miner] || Application to search for short sequence motifs in Proteins. <br />
|}<br />
<br />
<br />
==Upgrading MediaWiki==<br />
<br />
<big>Step 1</big> '''- [http://www.mediawiki.org/wiki/Manual:Upgrading download latest stable version of MediaWiki]'''<br />
<br />
<br />
<big>Step 2</big> '''- determine database credentials from dreamhost'''<br />
<br />
;Get Hostname (e.g.):<br />
* mysql.bradleymonk.com<br />
;Get Database name (e.g.):<br />
* bradleymonk_com<br />
;Get Database User name (e.g.):<br />
* username<br />
;Get Database User pass (e.g.):<br />
* password<br />
<br />
<br />
<big>Step 3</big> '''- back-up existing files and the database'''<br />
<br />
*Go to: mysql.example.com<br />
*Enter db user and password when prompted<br />
*click on db in left panel<br />
*click on the "Export" tab<br />
*check "Save as file"<br />
*click "Go"<br />
<br />
<br />
<big>Step 4</big> '''- FTP the <code>mediawiki.tar.gz</code> file to the /w directory'''<br />
<br />
<br />
<big>Step 5</big> '''- Unpack the tarball'''<br />
<br />
;Use the console to SSH into the /w directory and unpack tarball<br />
* <code>$ssh bradleymonk.com -l username</code><br />
* Change to the /w directory<br />
:* <code>$tar xvzf mediawiki.tar.gz</code><br />
*this unpacks into a new folder: <code>/w/mediawiki</code><br />
<br />
<br />
<big>Step 6</big> '''- Move files and rename directory'''<br />
<br />
;Copy files from old to new directory<br />
* LocalSettings.php<br />
* logofile.png<br />
* images (or upload) directory<br />
* extension directory<br />
* custom skins<br />
* .htaccess file<br />
<br />
;Swap-rename directories<br />
*rename old directory <code>/w_old</code><br />
*move new directory out of <code>/w_old</code> to top level domain<br />
*rename new directory to <code>/w</code><br />
<br />
<br />
<big>Step 7</big> '''- Run MediaWiki update script'''<br />
<br />
* login with SSH again:<br />
:* <code>$ssh bradleymonk.com -l username</code><br />
* change to the <code>/w/maintenance</code> directory<br />
* enter: <code>$php update.php</code><br />
<br />
<br />
* If the prompt gives an error like: "you're using php version 2.x mediawiki requires version 3.x"<br />
* try entering these:<br />
:* <code>$/usr/local/php53/bin/php update.php</code><br />
:* <code>$/usr/local/php54/bin/php update.php</code><br />
<br />
<br />
;Check if the upgrade worked by going to the MediaWiki page "Special Version"<br />
<br />
== Quoting ==<br />
{{cquote|SAN DIEGO -- Money -- does it draw students to their future careers? The answer is no, according to statistics and students at San Diego State University.}}<br />
<br />
<pre><br />
{{cquote|quote goes here}}<br />
</pre><br />
<br />
<br />
==MediaWiki Tips==<br />
<br />
*To remove the arrow icon from external links, navigate to MediaWiki:Common.css and add this line of code<br />
<nowiki><br />
#content a[href ^="http://"].external {background: center right no-repeat; padding-right: 1px;}<br />
</nowiki><br />
<br />
<br />
==Button Template==<br />
<br />
<nowiki>{{Button|TPB=.5em| Button Text }}</nowiki><br />
<br />
{{Button|TPB=.5em| Button Text }}<br />
<br />
<br />
==Table Map==<br />
<br />
See here: [[Table Map]]<br />
<br />
<br />
<br />
== Labeled Section Transclusion ==<br />
<br />
<big>See: [http://www.mediawiki.org/wiki/Extension:Labeled_Section_Transclusion Extension:Labeled_Section_Transclusion]</big><br />
<br />
=== Step 1: Mark off sections ===<br />
<br />
Mark off sections in the text using <nowiki><section></nowiki></> tags like this:<br />
<br />
<nowiki><br />
<section begin=Hayashi_Malinow_2000_Science /><br />
<br />
{{Article|Hayashi Malinow|2000|Science • PDF|10731148|Driving AMPA receptors into synapses}}<br />
<br />
<section end=Hayashi_Malinow_2000_Science /><br />
</nowiki><br />
<br />
Note that this uses two individual markers, rather than normal XML open/close tags, which simplifies nested or overlapping sections. This allows you to insert section tags without worrying about interfering with other sections.<br />
<br />
=== Step 2: Transclude the section ===<br />
<br />
Call the parser function to transclude it, i.e. to transclude a section called ''chapter1'' from a page called ''[[Articles]]'':<br />
<br />
<nowiki><br />
{{#section:Articles|Hayashi_Malinow_2000_Science}}<br />
</nowiki><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Category:Templates]]</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Help:Contents&diff=3917Help:Contents2020-05-04T11:34:36Z<p>Monakhos: </p>
<hr />
<div>{{Box|width=255px|MediaWiki [[Help]] Pages|<br />
* [[http://en.wikipedia.org/wiki/Help:Wiki_markup MediaWiki Markup]]<br />
* [[:Category:Templates]]<br />
* [http://www.onesci.com/Help:Contents OneSci Help]<br />
* [http://en.wikipedia.org/wiki/Help:Wiki_markup#Images Mediawiki Markup Images]<br />
* [http://www.mediawiki.org/wiki/Help:Magic_words MediaWiki Magic Words]<br />
* [http://www.mediawiki.org/wiki/Help:Tables MediaWiki Tables]<br />
* [http://commons.wikimedia.org/wiki/Help:Gadget-ImageAnnotator/Installation MediaWiki ImageAnnotator]<br />
* [http://www.mediawiki.org/wiki/Extension:Cite/Cite.php#Separating_references_from_text Mediawiki References]<br />
* [http://www.mediawiki.org/wiki/Manual:Math Math]<br />
* [http://en.wikipedia.org/wiki/Help:Template Template Tips]<br />
* [http://meta.wikimedia.org/wiki/Help:Advanced_templates Advanced Templates]<br />
* [http://en.wikipedia.org/wiki/Wikipedia:Transclusion Transclusion]<br />
}}<br />
<br />
{{Box|width=255px|Bradleymonk.com .JS|<br />
*[[MediaWiki:Common.js]]<br />
*[[MediaWiki:LAPI.js]]<br />
*[[MediaWiki:Gadget-ImageAnnotator.js]]<br />
*[[MediaWiki:ImageAnnotatorConfig.js]]<br />
*[[MediaWiki:LAPI.js]]<br />
*[[MediaWiki:TextCleaner.js]]<br />
*[[MediaWiki:Tooltips.js]]<br />
*[[MediaWiki:UIElements.js]]<br />
*[[MediaWiki:AjaxSubmit.js]]<br />
}}<br />
<br />
{{Box|width=255px|HTML & CSS|<br />
* [https://www.mediawiki.org/wiki/Manual:CSS Manual:CSS]<br />
*: [[MediaWiki:Common.css]]<br />
* [https://www.mediawiki.org/wiki/Manual:Skin_configuration Manual:Skin_configuration]<br />
* [https://www.mediawiki.org/wiki/Manual:Skinning Manual:Skinning]<br />
* [https://www.mediawiki.org/wiki/Help:Skins Help:Skins]<br />
}}<br />
<br />
<br />
{{Clear}}<br />
<br />
<br />
==Lua Scripting==<br />
<br />
* [https://www.mediawiki.org/wiki/Extension:Scribunto Mediawiki Extension:Scribunto]<br />
<br />
* Latest Update: [http://bradleymonk.com/Module:Yesno Modules still not working] < this is a medium-priority TBD when time permits<br />
<br />
== Adding HTML5 Video to MediaWiki ==<br />
I've enabled three options for embedding animated video in this wiki. '''Option 1: PopFig video''' will embed the video a popup box. The video is hidden in a semantic mediawiki icon; mouseover of this icon will pop-open the video. This option makes use of a semantic mediawiki parser tag, secure html extension, and several layers of transclusion. '''Option 2: mediaplayer video''' will embed an inline video using the <code><nowiki><mediaplayer></nowiki></code> tag. The option doesn't require anything but a stand-alone extension. '''Option 3: Widget:Html5media''' makes use of of an Html5 widget. This option is good for very small animations, but does has a fixed size so the portion of a larger videos that extends beyond ~200 pixels will be hidden.<br />
----<br />
<br />
=== Option 1: PopFig ===<br />
<br> {{PopFig|[[File:Dot.png]]|<html><br />
<video src="http://bradleymonk.com/media2/EXAMPLE.mov" controls></video> <br> <br />
<div style='color:white; width:400px'>This is an example of a movie caption. The text will automatically wrap</div><br />
</html>||<big>SI video1 </big>}}<br />
<br />
{{Style|class=div|size=90%|align=left|font=courier|background=#EBFAEB|color=black|pad=8px|margin=6px|<br />
<nowiki> <br />
{{PopFig|[[File:Dot.png]]|<html><br />
<br />
<video src="http://bradleymonk.com/media2/EXAMPLE.mov" controls></video> <br> <br />
<br />
<div style='color:white; width:400px'>This is an example of a movie caption. The text will automatically wrap</div><br />
<br />
</html>||<big>SI video1 </big>}}<br />
</nowiki> <br />
}}<br />
----<br />
<br />
=== Option 2: mediaplayer ===<br />
<br> <mediaplayer image='http://bradleymonk.com/media2/EXAMPLE.png' width='500' height='300'>http://bradleymonk.com/media2/EXAMPLE.mov</mediaplayer><br />
<br />
{{Style|class=div|size=90%|align=left|font=courier|background=#EBFAEB|color=black|pad=8px|margin=6px|<br />
<nowiki> <br />
<mediaplayer image='http://bradleymonk.com/media2/EXAMPLE.png' width='500' height='300'>http://bradleymonk.com/media2/EXAMPLE.mov</mediaplayer> <br />
</nowiki><br />
}}<br />
<br />
If the video is uploaded using the native wiki upload link in the sidebar, this option can be employed simply by pasting the [[Example uploaded video|uploaded video]] file name between the <code><nowiki><mediaplayer> </mediaplayer></nowiki></code> tags like so:<br />
<br />
{{Style|class=div|size=90%|align=left|font=courier|background=#EBFAEB|color=black|pad=8px|margin=6px|<br />
<nowiki> <br />
<mediaplayer image='http://www.bradleymonk.com/w/images/8/82/RenderTorus.jpg' width='500' height='300'>File:RenderTorus.mp4</mediaplayer><br />
</nowiki><br />
}}<br />
<br />
<br />
<mediaplayer image='http://www.bradleymonk.com/w/images/8/82/RenderTorus.jpg' width='500' height='300'>File:RenderTorus.mp4</mediaplayer><br />
----<br />
<br />
=== Option 3: [[Widget:Html5media|Widget:Html5media]] ===<br />
<br> {{Box|font=90%|width=45%|float=left|text=12px|boarder=solid #aaa 1px|Embeded Version|<br />
<br />
{{#widget:Html5media<br />
|url=http://bradleymonk.com/media2/DIFFUSE.mp4<br />
|width=200<br />
|height=200<br />
}}<br />
<br />
<pre><br />
{{#widget:Html5media<br />
|url=http://bradleymonk.com/media2/DIFFUSE.mp4<br />
|width=200<br />
|height=200<br />
}}<br />
</pre><br />
<br />
}}<!-- END BOX --><br />
<br />
{{Box|font=120%|width=45%|float=left|text=12px|boarder=solid #aaa 1px|Popup Version|<br />
<br />
Hover mouse over icon to open: {{Popup|{{#widget:Html5media|url=http://bradleymonk.com/media2/DIFFUSE.mp4|width=200|height=200}}}}<br />
<br />
<br />
<pre><br />
{{Popup|{{#widget:Html5media<br />
|url=http://bradleymonk.com/media2/DIFFUSE.mp4<br />
|width=200<br />
|height=200<br />
}}}}<br />
</pre><br />
<br />
}}<!-- END BOX --><br />
<br />
{{Clear}}<br />
<br />
==Semantic Mediawiki Popups==<br />
<br />
<big>[http://semantic-mediawiki.org/wiki/Help:Adding_tooltips Semantic Mediawiki Tool Tips]</big><br />
<br />
The parser function '''#info''' allows to add tooltips containing additional information or warnings.<br />
<br />
<big>Syntax</big><br><br />
<br />
<br />
* <code><nowiki>{{#info: <text> | <icon> }}</nowiki></code><br />
<br />
* you must include the "nowiki" tag around any equal signs if you want to use styles inside the into popup <br />
** example: <code><nowiki><span style</nowiki><nowiki><nowiki>=</nowiki></nowiki><nowiki>"color:red"></nowiki></code> <br />
** result {{#info:text <span style<nowiki>=</nowiki>"color:red">this is red</span> text|warning}} <br />
* <code><nowiki>{{#info:text <span style<nowiki>=</nowiki>"color:red">this is red</span> text}}</nowiki></code><br />
<br />
<br />
* If you want to have a large image popup, the best way to do this is using these inline styles with the span tag<br />
** <code>position:fixed; left:10px; top:10px; overflow:visible;</code><br />
** inline text {{#info:text <span style<nowiki>=</nowiki>"position:fixed; left:10px; top:10px; overflow:visible;">THIS WILL PUT THE IMAGE UP HERE</span> text}} inline text<br />
** Use the Popupimg template inside the Popup template to do exactly this<br />
<br />
<!--<br />
** example: <code><nowiki>{{#info: {{Popupimg|[[File:Nicoll2011 Fig1.png]]}} }}</nowiki></code><br />
** result: {{#info: {{Popupimg|[[File:Nicoll2011 Fig1.png]]}} }}<br />
** example: <code><nowiki>{{Popup| {{Popupimg|[[File:Nicoll2011 Fig1.png]] }} }}</nowiki></code><br />
** result: {{Popup| {{Popupimg|[[File:Nicoll2011 Fig1.png]]}} }}<br />
--><br />
<br />
==Extension LinkTitles==<br />
<br />
;AUTO GENERATED SITE LINKS<br />
<br />
* [http://www.mediawiki.org/wiki/Extension:LinkTitles MediaWiki Extension Page]<br />
<br />
<pre><br />
require_once( "$IP/extensions/LinkTitles/LinkTitles.php" );<br />
$wgLinkTitlesSmartMode = false; // false = case sensitive<br />
$wgLinkTitlesMinimumTitleLength = 7; // default is 3<br />
$wgLinkTitlesSkipTemplates = true; // skip any text in a {{template|text}}<br />
$wgLinkTitlesFirstOnly = true; // only link the first occurrence<br />
$wgLinkTitlesBlackList = array();<br />
$wgLinkTitlesBlackList[] = 'MediaWiki';<br />
$wgLinkTitlesBlackList[] = 'mediaplayer';<br />
</pre><br />
<br />
; USE THE __NOAUTOLINKS__ TAG ON A WIKI PAGE TO PREVENT LINKING ON THAT PAGE<br />
<br />
<pre><br />
__NOAUTOLINKS__ <br />
</pre><br />
<br />
__NOAUTOLINKS__<br />
<br />
==Template:SlideBox==<br />
<nowiki><br />
{{SlideBox|width=80%|float=left|<br />
Header [[Content]] Is Shown<br />
|<br />
Body text is hidden<br />
}}<br />
</nowiki><br />
<br />
==Customize Editing Toolbar==<br />
http://bradleymonk.com/User:Bradley_Monk/common.js<br />
http://www.mediawiki.org/wiki/Extension:WikiEditor/Toolbar_customization<br />
<br />
;section<br />
*{String} (optional) The name of the section in the WikiEditor. Defaults to 'main'<br />
;id<br />
*{String} (required) Unique id (ie. 'my-button')<br />
;icon<br />
*{String} (recommended) URL to the icon, should be square about 21 to 22px<br />
;label<br />
*{String} (required) Tooltip displayed when hovering button<br />
;insertBefore<br />
*{String} (optional) Wikitext to be inserted before the cursor on-click<br />
;sampleText<br />
*{String} (optional) Text inserted in place of the cursor if no text was selected<br />
;insertAfter<br />
*{String} (optional) Wikitext to be inserted after the cursor on-click<br />
;callback<br />
*{Function} (optional) Called when the button is clicked<br />
;autoSummary<br />
*{mixed} (optional) Null or an Object with the following properties:<br />
**summary: {String} (required) Edit summary that should be used<br />
**position: {String} (optional) 'append', 'prepend' or 'replace'<br />
**delimiter: {String} (optional) delimiter between the (possibly) current summary and the to-be-inserted summary<br />
<br />
==Syntax Highlighting==<br />
The '''Extension:SyntaxHighlight GeSHi''' tag displays formatted [[w:source code|source code]] with the <code>&lt;syntaxhighlight&gt;</code> tag.<br />
<br />
This extension also adds coloring according to the code language settings. Like the <code>&lt;pre&gt;</code> tags and the <code>&lt;[[Extension:Poem|poem]]&gt;</code> tags, the tags shows the coding exactly as it was typed, preserving white space. <br />
<br />
This extension also can create line numbers.<br />
<br />
=== Usage ===<br />
On the wiki page, you can now use "syntaxhighlight" elements:<br />
<br />
<pre><nowiki><br />
<syntaxhighlight lang="php"><br />
<?php<br />
$v = "string"; // sample initialization<br />
?><br />
html text<br />
<?<br />
echo $v; // end of php code<br />
?><br />
</syntaxhighlight><br />
</nowiki></pre><br />
<br />
shows:<br />
<syntaxhighlight lang="php"><br />
<?php<br />
$v = "string"; // sample initialization<br />
?><br />
html text<br />
<?<br />
echo $v; // end of php code<br />
?><br />
</syntaxhighlight><br />
<br />
=== Alternative &lt;source&gt; tag ===<br />
Before [[rev:50696]], the extension used the tag <code>[[Extension:SyntaxHighlighter|&lt;source&gt;]]</code>.<br />
This is still supported, but <code>&lt;syntaxhighlight&gt;</code> avoids conflicts if your source code itself contains <nowiki><source></nowiki> (for example XML).<br />
<br />
=== Parameters ===<br />
; <code>lang="??"</code><br />
: Defines what programming language the source code is using. This affects how the extension highlights the source code. See the section "[[#Supported_languages|Supported languages]]" in this page for details of supported languages.<br />
; <code>line="GESHI_NORMAL_LINE_NUMBERS|GESHI_FANCY_LINE_NUMBERS"</code><br />
: Type of line numbering to use. If you do not provide this parameter, then lines will not be numbered. Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#enabling-line-numbers enable_line_numbers] the flag in GeSHi.<br />
; <code>line</code><br />
: Equivalent to <code>'''line="GESHI_FANCY_LINE_NUMBERS"</code><br />
; <code>line start="??"</code><br />
: Use together with the parameter "'''line'''". Define the start number of the line. If you type line start="55", it will start counting at 55, then 56,57,58... and so on. Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#starting-line-numbers start_line_numbers_at] method on GeSHi<br />
<!--<br />
; <code>case</code><br />
: Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#auto-caps-nocaps set_case_keywords] method on GeSHi<br />
; <code>tab</code><br />
: Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#setting-tab-width set_tab_width] method on GeSHi<br />
--><br />
; <code>highlight="??"</code><br />
: Specifies which line is highlighted. Note that the parameter '''line start="??"''' doesn't affect how it counts the lines. <br />
; <code>enclose="??"</code><br />
: Specifies what container is used to enclose the source code. Takes values "pre" (default value), "div", "none". Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#the-code-container set_header_type] method on GeSHi. Choosing "div" will cause text to wrap, which is helpful if text is extending off the edge of the screen, causing horizontal scrolling.<br />
; <code>strict</code><br />
: Type the name of the parameter to enable the strict mode. Corresponds to [http://qbnz.com/highlighter/geshi-doc.html#using-strict-mode enable_strict_mode] method on GeSHi.<br />
<br />
The effect and usage of these parameters can be consulted in [http://qbnz.com/highlighter/geshi-doc.html GeSHi's documentation].<br />
<br />
Since r22246, you can override the colors using '''MediaWiki:Geshi.css'''.<br />
<br />
=== More Usage ===<br />
When line numbering is added with ''line'', long code lines will be wrapped. See the example below. When ''text'' is the selected language, and numbering is used, the behaviour resembles the use of ''pre'' tags with numbering and long-line wrapping.<br />
<br />
The following example shows how to color an HTML code listing:<br />
<pre><syntaxhighlight lang="html4strict" line start="100" highlight="5" enclose="div"><br />
HTML module goes here...<br />
</syntaxhighlight><br />
</pre> <br />
A typical result is just: <br />
<syntaxhighlight lang="html4strict" line start="100" highlight="5" enclose="div"><br />
<!--This is a comment. Comments are not displayed in the browser--><br />
<table align=center style="background: ivory;color:maroon;font-style:italic;font-family:arial;font-weight:bold;font-size:10pt;"> <br />
<tr><th> Heading 1 </th><th> Heading 2 </th></tr><br />
<tr><br />
<td style="padding:10px;"> This is cell 1 text </td><br />
<td style="padding:10px;"> This is cell 2 text </td><br />
</tr><br />
</table><br />
</syntaxhighlight><br />
<br />
=== Supported languages ===<br />
These are the languages known by GeSHi that can be used in the '''lang''' parameter.<br />
<br />
<small>Note that installed GeSHi version (can be revealed by previewing e.g. <code>&lt;syntaxhighlight lang="-"&gt;&lt;/syntaxhighlight&gt;</code>) might not be the most recent version; see [[bugzilla:10967]] for Wikimedia sites.</small><br />
<br />
<small>The GeSHi sources for each language can be found in [http://sourceforge.net/p/geshi/code/HEAD/tree/trunk/geshi-1.0.X/src/geshi/ VCS on SourceForge]. Remember to go and select the version that the MediaWiki installation uses.</small><br />
<br />
{| class="wikitable" style="float:left"<br />
!Code||Language<br />
|-<br />
|<code>abap</code>||[[:en:ABAP|ABAP]]<br />
|-<br />
|<code>actionscript</code>||[[:en:ActionScript|ActionScript]]<br />
|-<br />
|<code>ada</code>||[[:en:Ada (programming language)|Ada]]<br />
|-<br />
|<code>apache</code>||[[:en:Apache HTTP Server|Apache Configuration]]<br />
|-<br />
|<code>applescript</code>||[[:en:AppleScript|AppleScript]]<br />
|-<br />
|<code>asm</code>||[[:en:Assembly language|Assembly]]<br />
|-<br />
|<code>asp</code>||[[:en:Active Server Pages|Active Server Pages (ASP)]]<br />
|-<br />
|<code>autoit</code>||[[:en:AutoIt|AutoIt]]<br />
|-<br />
|<code>bash</code>||[[:en:Bash (Unix shell)|Bash]]<br />
|-<br />
|<code>basic4gl</code>||[[:en:Basic4GL|Basic4GL]]<br />
|-<br />
|<code>bf</code>||[[:en:Brainfuck|Brainfuck]]<br />
|-<br />
|<code>blitzbasic</code>||[[:en:Blitz BASIC|Blitz BASIC]]<br />
|-<br />
|<code>bnf</code>||[[:en:Backus-Naur Form|Backus-Naur Form]]<br />
|-<br />
|<code>c</code>||[[:en:C (programming language)|C]]<br />
|-<br />
|<code>c_mac</code>||C (Mac)<br />
|-<br />
|<code>caddcl</code>||[[:en:Dialog Control Language|AutoCAD DCL]]<br />
|-<br />
|<code>cadlisp</code>||[[:en:AutoLISP|AutoLISP]]<br />
|-<br />
|<code>cfdg</code>||CFDG<br />
|-<br />
|<code>cfm</code>||[[:en:ColdFusion_Markup_Language|ColdFusion Markup Language]]<br />
|-<br />
|<code>cil</code>||[[:en:Common_Intermediate_Language|Common Intermediate Language (CIL)]]<br />
|-<br />
|<code>cobol</code>||[[:en:COBOL|COBOL]]<br />
|-<br />
|<code>cpp-qt</code>||[[:en:Qt (toolkit)|C++ (Qt toolkit)]]<br />
|-<br />
|<code>cpp</code>||[[:en:C++|C++]]<br />
|-<br />
|<code>csharp</code>||[[:en:C Sharp (programming language)|C#]]<br />
|-<br />
|<code>css</code>||[[:en:Cascading Style Sheets|Cascading Style Sheets (CSS)]]<br />
|-<br />
|<code>d</code>||[[:en:D (programming language)|D]]<br />
|-<br />
|<code>delphi</code>||[[:en:Delphi programming language|Delphi]]<br />
|-<br />
|<code>diff</code>||[[:en:diff|Diff]]<br />
|-<br />
|<code>div</code>||DIV<br />
|-<br />
|<code>dos</code>||[[:en:DOS batch file|DOS batch file]]<br />
|-<br />
|<code>dot</code>||[[:en:DOT language|DOT]]<br />
|-<br />
|<code>eiffel</code>||[[:en:Eiffel (programming language)|Eiffel]]<br />
|-<br />
|<code>fortran</code>||[[:en:Fortran|Fortran]]<br />
|-<br />
|<code>freebasic</code>||[[:en:FreeBASIC|FreeBASIC]]<br />
|-<br />
|<code>gambas</code>||[[:en:Gambas_programming_language|Gambas]]<br />
|-<br />
|<code>genero</code>||Genero<br />
|-<br />
|<code>gettext</code>||[[:en:GNU_gettext|GNU internationalization (i18n) library]]<br />
|-<br />
|<code>glsl</code>||[[:en:GLSL|OpenGL Shading Language (GLSL)]]<br />
|-<br />
|<code>gml</code>||[[:en:Game Maker Language|Game Maker Language (GML)]]<br />
|-<br />
|<code>gnuplot</code>||[[:en:Gnuplot|gnuplot]]<br />
|-<br />
|<code>groovy</code>||[[:en:Groovy (programming language)|Groovy]]<br />
|-<br />
|<code>haskell</code>||[[:en:Haskell (programming language)|Haskell]]<br />
|-<br />
|<code>hq9plus</code>||HQ9+<br />
|-<br />
|<code>html4strict</code>||[[:en:HTML|HTML]]<br />
|-<br />
|<code>html5</code>||[[:en:HTML5|HTML5]]<br />
|-<br />
|<code>idl</code>||[[:en:Universal Network Objects|Uno IDL]]<br />
|-<br />
|<code>ini</code>||[[:en:INI file|INI]]<br />
|-<br />
|<code>inno</code>||[[:en:Inno Setup|Inno]]<br />
|-<br />
|<code>intercal</code>||[[:en:INTERCAL|INTERCAL]]<br />
|-<br />
|<code>io</code>||[[:en:Io (programming language)|Io]]<br />
|-<br />
|<code>java</code>||[[:en:Java (programming language)|Java]]<br />
|-<br />
|<code>java5</code>||[[:en:Java (programming language)|Java(TM) 2 Platform Standard Edition 5.0 ]]<br />
|-<br />
|<code>javascript</code>||[[:en:JavaScript|JavaScript]]<br />
|-<br />
|<code>kixtart</code>||[[:en:KiXtart|KiXtart]]<br />
|-<br />
|<code>klonec</code>||Klone C<br />
|-<br />
|<code>klonecpp</code>||Klone C++<br />
|-<br />
|<code>latex</code>||[[:en:LaTeX|LaTeX]]<br />
|-<br />
|<code>lisp</code>||[[:en:Lisp (programming language)|Lisp]]<br />
|-<br />
|<code>lolcode</code>||[[:en:LOLCODE|LOLCODE]]<br />
|-<br />
|<code>lotusscript</code>||[[:en:LotusScript|LotusScript]]<br />
|-<br />
|<code>lua</code>||[[:en:Lua (programming language)|Lua]]<br />
|}<br />
{| class="wikitable" style="float:left"<br />
!Code||Language<br />
|-<br />
|<code>m68k</code>||[[:en:Motorola 68000|Motorola 68000 Assembler]]<br />
|-<br />
|<code>make</code>||[[:en:Make_(software)|make]]<br />
|-<br />
|<code>matlab</code>||[[:en:MATLAB|MATLAB M]]<br />
|-<br />
|<code>mirc</code>||[[:en:mIRC scripting language|mIRC scripting language]]<br />
|-<br />
|<code>mxml</code>||[[:en:MXML|MXML]]<br />
|-<br />
|<code>mpasm</code>||[[:en:PIC microcontroller|Microchip Assembler]]<br />
|-<br />
|<code>mysql</code>||[[:en:MySQL|MySQL]]<br />
|-<br />
|<code>nsis</code>||[[:en:Nullsoft Scriptable Install System|Nullsoft Scriptable Install System (NSIS)]]<br />
|-<br />
|<code>objc</code>||[[:en:Objective-C|Objective-C]]<br />
|-<br />
|<code>ocaml-brief</code>||[[:en:Objective Caml|OCaml]]<br />
|-<br />
|<code>ocaml</code>||[[:en:Objective Caml|OCaml]]<br />
|-<br />
|<code>oobas</code>||[[:en:StarOffice Basic|OpenOffice.org Basic]]<br />
|-<br />
|<code>oracle8</code>||[[:en:PL/SQL|Oracle 8 SQL]]<br />
|-<br />
|<code>oracle11</code>||[[:en:PL/SQL|Oracle 11 SQL]]<br />
|-<br />
|<code>pascal</code>||[[:en:Pascal (programming language)|Pascal]]<br />
|-<br />
|<code>per</code>||per<br />
|-<br />
|<code>perl</code>||[[:en:Perl|Perl]]<br />
|-<br />
|<code>php-brief</code>||[[:en:PHP|PHP]]<br />
|-<br />
|<code>php</code>||[[:en:PHP|PHP]]<br />
|-<br />
|<code>pixelbender</code>||[[:en:Adobe_Pixel_Bender|Pixel Bender]]<br />
|-<br />
|<code>plsql</code>||[[:en:PL/SQL|PL/SQL]]<br />
|-<br />
|<code>povray</code>||[[:en:POV-Ray|Persistence of Vision Raytracer]]<br />
|-<br />
|<code>powershell</code>||[[:en:Windows_PowerShell|Windows PowerShell]]<br />
|-<br />
|<code>progress</code>||[[:en:OpenEdge_Advanced_Business_Language|OpenEdge Advanced Business Language]]<br />
|-<br />
|<code>prolog</code>||[[:en:Prolog|Prolog]]<br />
|-<br />
|<code>providex</code>||[[:en:ProvideX|ProvideX]]<br />
|-<br />
|<code>python</code>||[[:en:Python (programming language)|Python]]<br />
|-<br />
|<code>qbasic</code>||[[:en:QBasic|QBasic/QuickBASIC]]<br />
|-<br />
|<code>rails</code>||[[:en:Ruby on Rails|Rails]]<br />
|-<br />
|<code>reg</code>||[[:en:Windows Registry|Windows Registry]]<br />
|-<br />
|<code>robots</code>||[[:en:Robots Exclusion Standard|robots.txt]]<br />
|-<br />
|<code>rsplus</code>||[[:en:R (programming language)|R]]<br />
|-<br />
|<code>ruby</code>||[[:en:Ruby (programming language)|Ruby]]<br />
|-<br />
|<code>sas</code>||[[:en:SAS System|SAS]]<br />
|-<br />
|<code>scala</code>||[[:en:Scala_(programming_language)|Scala]]<br />
|-<br />
|<code>scheme</code>||[[:en:Scheme (programming language)|Scheme]]<br />
|-<br />
|<code>scilab</code>||[[:en:Scilab|Scilab]]<br />
|-<br />
|<code>sdlbasic</code>||[[:en:SdlBasic|SdlBasic]]<br />
|-<br />
|<code>smalltalk</code>||[[:en:Smalltalk|Smalltalk]]<br />
|-<br />
|<code>smarty</code>||[[:en:Smarty|Smarty]]<br />
|-<br />
|<code>sql</code>||[[:en:SQL|SQL]]<br />
|-<br />
|<code>tcl</code>||[[:en:Tcl|Tcl]]<br />
|-<br />
|<code>teraterm</code>||[[:en:TeraTerm|Tera Term]]<br />
|-<br />
|<code>text</code>||[[:en:Plain text|Plain text]]<br />
|-<br />
|<code>thinbasic</code>||[[:en:thinBasic|thinBasic]]<br />
|-<br />
|<code>tsql</code>||[[:en:Transact-SQL|Transact-SQL]]<br />
|-<br />
|<code>typoscript</code>||[[:en:TYPO3|TypoScript]]<br />
|-<br />
|<code>vala</code>||[[:en:Vala_(programming_language)|Vala]]<br />
|-<br />
|<code>vb</code>||[[:en:Visual Basic|Visual Basic]]<br />
|-<br />
|<code>vbnet</code>||[[:en:Visual Basic .NET|Visual Basic .NET]]<br />
|-<br />
|<code>verilog</code>||[[:en:Verilog|Verilog]]<br />
|-<br />
|<code>vhdl</code>||[[:en:VHSIC Hardware Description Language|VHDL]]<br />
|-<br />
|<code>vim</code>||[[:en:Vimscript|Vimscript]]<br />
|-<br />
|<code>visualfoxpro</code>||[[:en:Visual FoxPro|Visual FoxPro]]<br />
|-<br />
|<code>visualprolog</code>||[[:en:Visual_Prolog|Visual Prolog]]<br />
|-<br />
|<code>whitespace</code>||[[:en:Whitespace_(programming_language)|Whitespace]]<br />
|-<br />
|<code>winbatch</code>||[[:en:Winbatch|Winbatch]]<br />
|-<br />
|<code>xml</code>||[[:en:XML|XML]]<br />
|-<br />
|<code>xorg_conf</code>||[[:en:Xorg.conf|Xorg.conf]]<br />
|-<br />
|<code>xpp</code>||[[:en:Microsoft_Dynamics_AX|X++]]<br />
|-<br />
|<code>z80</code>||[[:en:Zilog Z80|ZiLOG Z80 Assembler]]<br />
|}<br />
<div style="clear:both"></div><br />
<br />
=== Extra features ===<br />
<br />
; Default Source Language<br />
<br />
Added in [[rev:50693]].<br />
<br />
If the site mainly quotes the source code of a specific programming language, it would be helpful to set a default language.<br />
<br />
To do so, add a new variable to LocalSettings.php, just after the require_once line. We set [[:en:C (programming language)|C programming language]] as an example:<br />
<syntaxhighlight lang="php"><br />
require_once("$IP/extensions/SyntaxHighlight_GeSHi/SyntaxHighlight_GeSHi.php");<br />
$wgSyntaxHighlightDefaultLang = "c";<br />
</syntaxhighlight><br />
<br />
==LaTeX Math==<br />
<br />
; NEW WAY<br />
<br />
<pre><tex>f{(x)} = {\sum \lambda \cdot \bar{y} \over \sum \lambda^{ \over 2 n} }</tex></pre><br />
<br />
:: <tex>f{(x)} = {\sum \lambda \cdot \bar{y} \over \sum \lambda^{ \over 2 n} }</tex><br />
<br />
; OLD WAY<br />
<br />
<pre><br />
<math> f(x, \mu, \sigma) = \frac{1}{\sigma \sqrt{2\pi} } e^{ -\frac{(x-\mu)^2}{2\sigma^2} } </math><br />
</pre><br />
<br />
::<math><br />
f(x, \mu, \sigma) = \frac{1}{\sigma \sqrt{2\pi} } e^{ -\frac{(x-\mu)^2}{2\sigma^2} }<br />
</math><br />
<br />
; {{TeX}} and HTML<br />
<br />
<big>[http://en.wikipedia.org/wiki/Table_of_mathematical_symbols LIST OF MATH SYMBOLS IN HTML MARKUP]</big><br><br />
<br />
{| class="wikitable"<br />
|-<br />
! {{TeX}} Syntax ([[#Forced_PNG_rendering|forcing PNG]])<br />
! {{TeX}} Rendering<br />
! HTML Syntax<br />
! HTML Rendering<br />
|-<br />
| <code><nowiki><math>\alpha</math></nowiki></code><br />
| <math>\alpha</math><br />
| <code><nowiki>{{math|<VAR>&amp;alpha;</VAR>}}</nowiki></code><br />
| {{math|<VAR>&alpha;</VAR>}}<br />
|-<br />
| <code><nowiki><math> f(x) = x^2\,</math></nowiki></code><br />
| <math> f(x) = x^2\,</math><br />
| <code><nowiki>{{math|''f''(<var>x</var>) {{=}} <var>x</var><sup>2</sup>}}</nowiki></code><br />
| {{math|''f''(<var>x</var>) {{=}} <var>x</var><sup>2</sup>}}<br />
|-<br />
| <code><nowiki><math>\sqrt{2}</math></nowiki></code><br />
| <math>\sqrt{2}</math><br />
| <code><nowiki>{{math|{{radical|2}}}}</nowiki></code><br />
| {{math|{{radical|2}}}}<br />
|-<br />
| <code><nowiki><math>\sqrt{1-e^2}</math></nowiki></code><br />
| <math>\sqrt{1-e^2}</math><br />
| <code><nowiki>{{math|{{radical|1 &amp;minus; ''e''&amp;sup2;}}}}</nowiki></code><br />
| {{math|{{radical|1 &minus; ''e''&sup2;}}}}<br />
|}<br />
<br />
The codes on the left produce the symbols on the right, but the latter can also be put directly in the wikitext, except for &lsquo;=&rsquo;.<br />
<br />
{| class="wikitable"<br />
|-<br />
! Syntax<br />
! Rendering<br />
|- valign="top"<br />
|<pre><nowiki>&amp;alpha; &amp;beta; &amp;gamma; &amp;delta; &amp;epsilon; &amp;zeta;<br />
&amp;eta; &amp;theta; &amp;iota; &amp;kappa; &amp;lambda; &amp;mu; &amp;nu;<br />
&amp;xi; &amp;omicron; &amp;pi; &amp;rho; &amp;sigma; &amp;sigmaf;<br />
&amp;tau; &amp;upsilon; &amp;phi; &amp;chi; &amp;psi; &amp;omega;<br />
&amp;Gamma; &amp;Delta; &amp;Theta; &amp;Lambda; &amp;Xi; &amp;Pi;<br />
&amp;Sigma; &amp;Phi; &amp;Psi; &amp;Omega;<br />
</nowiki></pre><br />
| style="texhtml" |α β γ δ ε ζ<br <br />
/>η θ ι κ λ μ ν<br <br />
/>ξ ο π ρ σ ς<br <br />
/>τ υ φ χ ψ ω<br<br />
/>Γ Δ Θ Λ Ξ Π<br<br />
/>Σ Φ Ψ Ω<br />
|- valign="top"<br />
| valign="middle" | <pre><nowiki>&amp;int; &amp;sum; &amp;prod; &amp;radic; &amp;minus; &amp;plusmn; &amp;infty;<br />
&amp;asymp; &amp;prop; {{=}} &amp;equiv; &amp;ne; &amp;le; &amp;ge; <br />
&amp;times; &amp;middot; &amp;divide; &amp;part; &amp;prime; &amp;Prime;<br />
&amp;nabla; &amp;permil; &amp;deg; &amp;there4; &amp;Oslash; &amp;oslash;<br />
&amp;isin; &amp;notin; <br />
&amp;cap; &amp;cup; &amp;sub; &amp;sup; &amp;sube; &amp;supe;<br />
&amp;not; &amp;and; &amp;or; &amp;exist; &amp;forall; <br />
&amp;rArr; &amp;hArr; &amp;rarr; &amp;harr; &amp;uarr; <br />
&amp;alefsym; - &amp;ndash; &amp;mdash; <br />
</nowiki></pre><br />
| style="texhtml" |∫ ∑ ∏ √ − ± ∞<br <br />
/>≈ ∝ = ≡ ≠ ≤ ≥<br <br />
/>× · ÷ ∂ ′ ″<br <br />
/>∇ ‰ ° ∴ Ø ø<br<br />
/>∈ ∉ ∩ ∪ ⊂ ⊃ ⊆ ⊇<br<br />
/>¬ ∧ ∨ ∃ ∀<br <br />
/>⇒ ⇔ → ↔ ↑<br <br />
/>ℵ - – —<br />
|}<br />
<br />
==Image Annoation==<br />
<br />
<nowiki><br />
<div class="wpImageAnnotatorEnable"><br />
<span class="wpImageAnnotatorPageName" style="display:none;">[[Sandbox]]</span><br />
<span class="wpImageAnnotatorFullName" style="display:none;">[[Sandbox]]</span><br />
<div class="wpImageAnnotatorFile">[[File:Foo.png]]</div><br />
<div style="display:none;"><div><div><!--IMAGE NOTES BELOW -->{{ImageNoteEnd|id=-1}}<br />
</div><br />
</nowiki><br />
<br />
==Livelets==<br />
*[http://www.mediawiki.org/wiki/Extension:Livelets Livelets]<br />
<br />
To create livelets, wrap any areas of the page in the #live parser-function.<br />
<br />
In the following example, a transclusion of the page called Template:MyVerySlowTable has been turned into a livelet:<br />
<br />
<nowiki><br />
{{#live: {{MyVerySlowTable}} }}<br />
</nowiki><br />
<br />
<br />
==References and Citations==<br />
<br />
===Using Native Ref Tags===<br />
In-text references make it easy to copy the text to another page; on the other hand, they make it hard to read. References containing a lot of data, quotes or elaborate citation templates can make up a significantly larger fraction of the source than the text that will actually be visible. To avoid this, recent versions of the extension allow moving some or all of the references into the <code>&lt;references /></code> section, to the place where they will actually appear to the reader. Thus,<br />
<br />
<blockquote><br />
<tt><br />
According to scientists, the Sun is pretty big.<font color=red>'''&lt;ref name="miller" />'''</font><br /><br />
The Moon, however, is not so big.<font color=red>'''&lt;ref name="smith" />'''</font><br /><br />
<br /><br />
&#61;=Notes==<br />
<br /><br />
<font color=red>'''&lt;references>'''<br /><br />
'''&lt;ref name="miller">'''E. Miller, ''The Sun'', (New York: Academic Press, 2005), 23-5.'''&lt;/ref>'''</br><br />
'''&lt;ref name="smith">'''R. Smith, "Size of the Moon", ''Scientific American'', 46 (April 1978): 44-6.'''&lt;/ref>'''<br /><br />
'''&lt;/references>'''</font><br />
</tt><br />
</blockquote><br />
<br />
will have the same output as the [[#Example|first example]] above, although the numbering and order of the references will not in general be the same.<br />
<br />
----<br />
==HTML ==<br />
<br />
===Example===<br />
<br />
<html><br />
<iframe src="http://www.w3schools.com/tags/tag_iframe.asp" height="100" width="400" frameborder="1" seamless="seamless" style="float:left"></iframe><br />
</html><br />
<br />
{{Clear}}<br />
{{Box|width=600px|iFrame Code|<br />
<code><br />
<nowiki><br />
<html><br />
<iframe src="http://www.w3schools.com/tags/tag_iframe.asp" <br />
height="300" width="350" frameborder="1" seamless="seamless" style="float:left"><br />
</iframe><br />
</html><br />
</nowiki><br />
</code><br />
}}<br />
<br />
{{Clear}}<br />
===iFrame Attributes===<br />
<br />
{| class="wikitable"<br />
|-<br />
! Attribute !! Value !! Description<br />
|-<br />
| align || left, right, top, middle, bottom || Specifies the alignment of an iframe according to surrounding elements<br />
|-<br />
| frameborder || 1, 0 || Specifies whether or not to display a border around an iframe<br />
|-<br />
| height || pixels || Specifies the height of an iframe<br />
|-<br />
| longdesc || URL || Specifies a page that contains a long description of the [[content]] of an iframe<br />
|-<br />
| marginheight || pixels || Specifies the top and bottom margins of the [[content]] of an iframe<br />
|-<br />
| marginwidth || pixels || Specifies the left and right margins of the [[content]] of an iframe<br />
|-<br />
| name || name || Specifies the name of an iframe<br />
|-<br />
| [[sandbox]] || "", allow-forms, allow-same-origin, allow-scripts, allow-top-navigation || Enables a set of extra restrictions for the [[content]] in the iframe<br />
|-<br />
| scrolling || yes, no, auto || Specifies whether or not to display scrollbars in an iframe<br />
|-<br />
| seamless || seamless || Specifies that the iframe should look like it is a part of the containing document<br />
|-<br />
| src || URL || Specifies the address of the document to embed in the iframe<br />
|-<br />
| srcdoc || HTML_code || Specifies the HTML [[content]] of the page to show in the iframe<br />
|-<br />
| width || pixels || Specifies the width of an iframe<br />
|}<br />
<br />
==Tables==<br />
<br />
===wikitable===<br />
{| class="wikitable" style="background-color:white;"<br />
|-<br />
! Link !! Description<br />
|-<br />
|[http://vcell.org/bionetgen/ BioNetGen] || A web-based tool for automatically generating a biochemical reaction networks from user-specified rules for biomolecular interactions. <br />
|-<br />
|[http://bionumbers.hms.harvard.edu/ B10NUMB3R5] || (BIONUMBERS) A database of useful biological numbers. <br />
|-<br />
|[http://www.bioinformaticsservices.com/ BioInformatics Services] || BioInformatics Services is a biotechnology consulting firm specializing in using computational biology to solve complex biological and data analysis problems. <br />
|-<br />
|[http://www.cellmigration.org/resource/modeling/model_resources.shtml#softwares cellmigrationgateway] || modeling software <br />
|-<br />
|[http://www.cellml.org/ CellML] || The CellML language is an open standard based on the XML markup language at the University of Auckland and affiliated research groups. <br />
|-<br />
|[https://sites.google.com/site/compcellbio/ CompCell Bio Web] || Development site for teaching modules on quantitative cell biology. <br />
|-<br />
|[http://doqcs.ncbs.res.in/ DOQCS] || Quantitative Cellular Signaling (DOQCS) is a repository of models of signaling pathways. <br />
|-<br />
|[http://www.e-cell.org/ecell/ E-Cell] || A Multi-Algorithm, Multi-Timescale Simulation Software Environment. <br />
|-<br />
|[http://gfit.sourceforge.net/ Gfit] || A program for global regression analysis of experimental data. <br />
|-<br />
|[http://www.kitware.com/ Kitware] || Professional Visualization Solutions, Tools and Support. <br />
|-<br />
|[http://www.mcell.cnl.salk.edu/ MCell] || General Monte Carlo Simulator of Cellular Microphysiology. <br />
|-<br />
|[http://mnm.engr.uconn.edu/MNM/SMSSearchServlet Minimotif Miner] || Application to search for short sequence motifs in Proteins. <br />
|}<br />
<br />
<br />
==Upgrading MediaWiki==<br />
<br />
<big>Step 1</big> '''- [http://www.mediawiki.org/wiki/Manual:Upgrading download latest stable version of MediaWiki]'''<br />
<br />
<br />
<big>Step 2</big> '''- determine database credentials from dreamhost'''<br />
<br />
;Get Hostname (e.g.):<br />
* mysql.bradleymonk.com<br />
;Get Database name (e.g.):<br />
* bradleymonk_com<br />
;Get Database User name (e.g.):<br />
* username<br />
;Get Database User pass (e.g.):<br />
* password<br />
<br />
<br />
<big>Step 3</big> '''- back-up existing files and the database'''<br />
<br />
*Go to: mysql.example.com<br />
*Enter db user and password when prompted<br />
*click on db in left panel<br />
*click on the "Export" tab<br />
*check "Save as file"<br />
*click "Go"<br />
<br />
<br />
<big>Step 4</big> '''- FTP the <code>mediawiki.tar.gz</code> file to the /w directory'''<br />
<br />
<br />
<big>Step 5</big> '''- Unpack the tarball'''<br />
<br />
;Use the console to SSH into the /w directory and unpack tarball<br />
* <code>$ssh bradleymonk.com -l username</code><br />
* Change to the /w directory<br />
:* <code>$tar xvzf mediawiki.tar.gz</code><br />
*this unpacks into a new folder: <code>/w/mediawiki</code><br />
<br />
<br />
<big>Step 6</big> '''- Move files and rename directory'''<br />
<br />
;Copy files from old to new directory<br />
* LocalSettings.php<br />
* logofile.png<br />
* images (or upload) directory<br />
* extension directory<br />
* custom skins<br />
* .htaccess file<br />
<br />
;Swap-rename directories<br />
*rename old directory <code>/w_old</code><br />
*move new directory out of <code>/w_old</code> to top level domain<br />
*rename new directory to <code>/w</code><br />
<br />
<br />
<big>Step 7</big> '''- Run MediaWiki update script'''<br />
<br />
* login with SSH again:<br />
:* <code>$ssh bradleymonk.com -l username</code><br />
* change to the <code>/w/maintenance</code> directory<br />
* enter: <code>$php update.php</code><br />
<br />
<br />
* If the prompt gives an error like: "you're using php version 2.x mediawiki requires version 3.x"<br />
* try entering these:<br />
:* <code>$/usr/local/php53/bin/php update.php</code><br />
:* <code>$/usr/local/php54/bin/php update.php</code><br />
<br />
<br />
;Check if the upgrade worked by going to the MediaWiki page "Special Version"<br />
<br />
== Quoting ==<br />
{{cquote|SAN DIEGO -- Money -- does it draw students to their future careers? The answer is no, according to statistics and students at San Diego State University.}}<br />
<br />
<pre><br />
{{cquote|quote goes here}}<br />
</pre><br />
<br />
<br />
==MediaWiki Tips==<br />
<br />
*To remove the arrow icon from external links, navigate to MediaWiki:Common.css and add this line of code<br />
<nowiki><br />
#content a[href ^="http://"].external {background: center right no-repeat; padding-right: 1px;}<br />
</nowiki><br />
<br />
<br />
==Button Template==<br />
<br />
<nowiki>{{Button|TPB=.5em| Button Text }}</nowiki><br />
<br />
{{Button|TPB=.5em| Button Text }}<br />
<br />
<br />
==Table Map==<br />
<br />
See here: [[Table Map]]<br />
<br />
<br />
<br />
== Labeled Section Transclusion ==<br />
<br />
<big>See: [http://www.mediawiki.org/wiki/Extension:Labeled_Section_Transclusion Extension:Labeled_Section_Transclusion]</big><br />
<br />
=== Step 1: Mark off sections ===<br />
<br />
Mark off sections in the text using <nowiki><section></nowiki></> tags like this:<br />
<br />
<nowiki><br />
<section begin=Hayashi_Malinow_2000_Science /><br />
<br />
{{Article|Hayashi Malinow|2000|Science • PDF|10731148|Driving AMPA receptors into synapses}}<br />
<br />
<section end=Hayashi_Malinow_2000_Science /><br />
</nowiki><br />
<br />
Note that this uses two individual markers, rather than normal XML open/close tags, which simplifies nested or overlapping sections. This allows you to insert section tags without worrying about interfering with other sections.<br />
<br />
=== Step 2: Transclude the section ===<br />
<br />
Call the parser function to transclude it, i.e. to transclude a section called ''chapter1'' from a page called ''[[Articles]]'':<br />
<br />
<nowiki><br />
{{#section:Articles|Hayashi_Malinow_2000_Science}}<br />
</nowiki><br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
<br />
[[Category:Templates]]</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:Box&diff=3916Template:Box2020-05-04T11:29:33Z<p>Monakhos: </p>
<hr />
<div><div style="plainlinks; font-size:{{{font|150%}}}; text-align:center; border:{{{border|solid #aaa 1px}}}; font-family: serif; margin:0 1em 1em 0; width:{{{width|50%}}}; padding:7px; float:{{{float|left}}}; min-width:{{{min-width|100px}}};"><br />
<span style="plainlinks;color:#2F4F4F;">{{{1}}}</span><br />
<div style="plainlinks; font-size:{{{text|12px}}}; text-align:left; font-family:Arial; border-top:1px solid #CCC; padding:{{{padding|5px 0px 0px 0px}}}; overflow:hidden;"><br />
{{{2}}}<br />
</div><br />
</div><noinclude><br />
{{Clear}}<br />
<br />
<br />
<br />
<code><nowiki><br />
{{Box|font=150%|width=50%|float=left|text=12px|boarder=solid #aaa 1px|<br />
Header Font|<br />
<br />
text text text<br />
<br />
}}<br />
</nowiki></code><br />
{{Clear}}<br />
<br />
<br />
<br />
{{Box|font=150%|width=50%|float=left|text=12px|boarder=solid #aaa 1px|<br />
Header Font|<br />
<br />
text text text<br />
<br />
}}</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=MediaWiki:Common.css&diff=3915MediaWiki:Common.css2020-05-04T11:26:43Z<p>Monakhos: </p>
<hr />
<div>/* CSS placed here will be applied to all skins */<br />
<br />
@import url( 'https://fonts.googleapis.com/css?family=Noto+Sans|Noto+Serif ')<br />
<br />
/* Changes the default font used for MediaWiki to Noto Sans (does not include headings or monospaced text): */<br />
body {<br />
font-family: "Noto Sans", sans-serif;<br />
}<br />
<br />
/* Changes the default font used for MediaWiki headings to Noto Serif: */<br />
#content h1, <br />
#content h2 {<br />
font-family: "Noto Serif", serif;<br />
}</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=MediaWiki:Common.css&diff=3914MediaWiki:Common.css2020-05-04T11:20:10Z<p>Monakhos: </p>
<hr />
<div>/* CSS placed here will be applied to all skins */<br />
#content a[href ^="http://"].external<br />
{<br />
background: center right no-repeat; padding-right: 1px;<br />
}<br />
<br />
<br />
@import url( 'https://fonts.googleapis.com/css?family=Noto+Sans|Noto+Serif ')<br />
<br />
/* Changes the default font used for MediaWiki to Noto Sans (does not include headings or monospaced text): */<br />
body {<br />
font-family: "Noto Sans", sans-serif;<br />
}<br />
<br />
/* Changes the default font used for MediaWiki headings to Noto Serif: */<br />
#content h1, <br />
#content h2 {<br />
font-family: "Noto Serif", serif;<br />
}</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Websites&diff=1208Websites2013-06-13T03:22:20Z<p>Monakhos: </p>
<hr />
<div>* [http://bradleymonk.com Bradley Monk Homepage]<br />
* [http://onesci.com OneSci]<br />
* [http://braininformatics.com Brain Informatics]<br />
* [http://newgnu.com NewGNU]<br />
* [http://researcharticlereview.com Research Article Review]<br />
* [http://thoughtpatent.com Thought Patent]<br />
* [http://theopensourcejournal.com The Open Source Journal]<br />
* [http://trekate.com Trekate]<br />
* [http://webaxon.com Web Axon]<br />
* [http://camk2.com CaMK2]<br />
* [http://scienceprotocol.com Science Protocol]<br />
* [http://otasushi.com Sushi Ota San Diego]</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Websites&diff=1207Websites2013-06-13T03:20:48Z<p>Monakhos: Created page with "* [http://bradleymonk.com Bradley Monk Homepage] * [http://onesci.com OneSci] * [http://braininformatics.com Brain Informatics] * [http://newgnu.com NewGNU] * [http://research..."</p>
<hr />
<div>* [http://bradleymonk.com Bradley Monk Homepage]<br />
* [http://onesci.com OneSci]<br />
* [http://braininformatics.com Brain Informatics]<br />
* [http://newgnu.com NewGNU]<br />
* [http://researcharticlereview.com Research Article Review]<br />
* [http://thoughtpatent.com Thought Patent]<br />
* [http://theopensourcejournal.com The Open Source Journal]<br />
* [http://trekate.com Trekate]<br />
* [http://webaxon.com Web Axon]<br />
* [http://camk2.com CaMK2]<br />
* [http://scienceprotocol.com Science Protocol]</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Main_Page&diff=1206Main Page2013-06-13T03:16:12Z<p>Monakhos: </p>
<hr />
<div>{{Box|width=48%|font-size=14px|[[Connectome|Brain Connectome and Functional Connectivity Project]]|<br />
A [[connectome]] is a comprehensive map of the neural networks within the [[brain]]. It details the [http://en.wikipedia.org/wiki/Efferent_nerve_fiber efferent] and afferent pathways within and between [[brain]] regions. Functional Connectivity refers to the function of a particular [[brain]] region and its information processing role within a distributed neural network. The goal of this project is to create a platform where users can jump into the [[connectome]] at any given [[brain]] region and visually navigate to upstream and downstream regions; along the way, users can learn about the functional role of each [[brain]] region. All information has been collected from empirical sources and scientific databases, in particular, the [http://atlas.brain-map.org Allan Brain Atlas]. <br />
[[File:Connectome.jpg|450px|link=Connectome]]<br />
}}<br />
<br />
{{Box|width=45%|float=right|font-size=14px|[[Brain Molecular Pathways|Brain Molecular Pathways Project]]|<br />
This project aims to provide annotated sets of [[Molecular Pathways|molecular pathways]] involved in neural plasticity underlying learning and memory systems. In general, biological pathways display the series of interactions among molecules resulting in functional changes within cells and neural networks. Currently there are large scale projects dedicated to amassing pathway evidence via high-throughput methods. The goal is to translate this unwieldy biopathway data from several [http://www.genome.jp/kegg/ empirical databases] into visually digestible material, by [[Molecular Pathways|characterizing]] the features of molecular cascades most sensitive to an ''event of interest'' (e.g. fear conditioning or amphetamine addiction).<br />
<br />
[[File:Molecular-pathway8.gif]]<br />
}}<br />
<br />
<br />
<br />
<br />
{{Box|width=48%|Welcome to the official wiki of Brad Monk|<br />
[[Hello]] and welcome to [[User:Monakhos|my wiki]]. This is where I stash random information and have every intention of linking it all together someday. I'm not sure why you're here.. maybe trying to find one of my other wiki projects '''[http://onesci.com OneSci Science News]''' or '''[http://ucsd.onesci.com UCSD Psych Grad wiki]'''? If you are so inclined, recent additions to this wiki can be found in the box on the right. For a non-curated glimpse of my activity you can check out the [[Special:RecentChanges|latest wiki updates]]. Older wiki [[content]] can be accessed using the <nowiki>[search box]</nowiki> or perusing [[Special:AllPages| all pages]]. If you would like to contact me, you can find this info on [http://bradleymonk.com my home page].<br />
}}<br />
<br />
<br />
{{Box|width=48%|Selected & New Pages|<br />
*[[Western Blot Protocol]]<br />
*[[Qual]]<br />
*[[Neuron Types]]<br />
*[[Quantum Dots]]<br />
*[[LTP]]<br />
*[[Hippocampus]]<br />
*[[AMPAR Tracking]]<br />
*[[User:Monakhos]]<br />
*[[Special:NewFiles]]<br />
*[[Special:NewPages]]<br />
*[[Neuroscience Tools]]<br />
*[[Websites]]<br />
}}<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=975Western Blot Protocol2013-05-15T23:27:53Z<p>Monakhos: /* BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x [http://shop.expedeon.com/products/71-RunBlue-LDS-Antioxidant-and-Reducer/32-RunBlue-LDS-Sample-Buffer-10ml-4x-Conc/ RunBlue] LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/XnEdmk1Sqvg" frameborder="0" allowfullscreen></iframe></html><br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Prepare [[Mini-PROTEAN]] [http://www.bio-rad.com/prd/en/US/LSR/PDP/5cf78e19-7ed5-4373-a988-3e62456a488e/Mini-PROTEAN-Tetra-Cell Tetra Cell] tank for electrophoresis (see video)<br />
*Use these gels if doing electrophoresis on the Mini-PROTEAN rig: 4–20% [[Mini-PROTEAN TGX]] Precast Gel [http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=456-1093 #456-1093]<br />
*Remove green tape strip from bottom of gel cassette<br />
*Set up gel cassette in Mini-PROTEAN gel holder, wells facing inward, and put in tank<br />
**If running only 1 or 2 gels remove the 2nd companion gel-holder<br />
*Remove comb from top of gel cassette<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment making sure RB covers wells <br />
*Fill exterior compartment with RB<br />
*Rinse wells with RB using pipette<br />
<br />
<br />
*Load [https://products.invitrogen.com/ivgn/product/LC5925 SeeBlue+2] [http://products.invitrogen.com/ivgn/product/LC5925 standard] into first and 5th lane<br />
**[[Mini-PROTEAN TGX]] Precast gels can only handle up to 30 μL of total solution<br />
**[[Mini-PROTEAN TGX]] Precast gels have 10 wells, plan accordingly<br />
*Load 25 μL sample into each gel well<br />
*record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/VgAuZ6dBOfs" frameborder="0" allowfullscreen></iframe></html><br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Use Immun-Blot PVDF Membrane [http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=162-0174 #162-0174]<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=MediaWiki:Common.js&diff=974MediaWiki:Common.js2013-05-15T23:17:30Z<p>Monakhos: </p>
<hr />
<div>/* Any JavaScript here will be loaded for all users on every page load. */<br />
<br />
if (wgNamespaceNumber != -1) importScript ('MediaWiki:Gadget-ImageAnnotator.js');<br />
<br />
/*importScriptURI('//toolserver.org/~dapete/ime/ime.js');*/</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Mini-PROTEAN_TGX&diff=973Mini-PROTEAN TGX2013-05-15T23:14:14Z<p>Monakhos: Created page with " *Mini-PROTEAN TGX, 4-20%, 10-well *[http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=456-1093 Link] *Supplier ..."</p>
<hr />
<div><br />
<br />
<br />
*Mini-PROTEAN TGX, 4-20%, 10-well<br />
*[http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=456-1093 Link]<br />
*Supplier Bio-Rad Laboratories<br />
*Part Number 4561093<br />
*Manufacturer Name Bio-Rad Laboratories - Life Sciences<br />
*Manufacturer Part Number 4561093<br />
*System Packaging EA<br />
*Supplier UOM EA<br />
*Supplier Size Pkg of 10<br />
*Additional Information MP-TGX, 4-20%, 10W, 30µl, 10 gels per box, 7.2 x 8.6 cm<br />
*Category Precast Acrylamide Gels<br />
*Category UNSPSC 41-10-53-20<br />
*Color <br />
*Hazardous Material Code 0<br />
*Height 2.750000004<br />
*Image URL http://www.bio-rad.com/webroot/web/... <br />
*Lead Time 1<br />
*Length 7.000000011<br />
*More Information URL http://www.bio-rad.com/prd/en/US/ad... <br />
*MSDS URL http://www.bio-rad.com/msds/ <br />
*Packaging Blue Ice<br />
*Price Code 1 1456<br />
*Price Code 2 CONS<br />
*Quantity 1<br />
*UNSPSC 41-10-53-20<br />
*Weight 1.329999907</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=972Western Blot Protocol2013-05-15T23:13:15Z<p>Monakhos: /* 3. SDS-PAGE (GEL ELECTROPHORESIS) */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x [http://shop.expedeon.com/products/71-RunBlue-LDS-Antioxidant-and-Reducer/32-RunBlue-LDS-Sample-Buffer-10ml-4x-Conc/ RunBlue] LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/XnEdmk1Sqvg" frameborder="0" allowfullscreen></iframe></html><br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Prepare [[Mini-PROTEAN]] [http://www.bio-rad.com/prd/en/US/LSR/PDP/5cf78e19-7ed5-4373-a988-3e62456a488e/Mini-PROTEAN-Tetra-Cell Tetra Cell] tank for electrophoresis (see video)<br />
*Use these gels if doing electrophoresis on the Mini-PROTEAN rig: 4–20% [[Mini-PROTEAN TGX]] Precast Gel [http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=456-1093 #456-1093]<br />
*Remove green tape strip from bottom of gel cassette<br />
*Set up gel cassette in Mini-PROTEAN gel holder, wells facing inward, and put in tank<br />
**If running only 1 or 2 gels remove the 2nd companion gel-holder<br />
*Remove comb from top of gel cassette<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment making sure RB covers wells <br />
*Fill exterior compartment with RB<br />
*Rinse wells with RB using pipette<br />
<br />
<br />
*Load [https://products.invitrogen.com/ivgn/product/LC5925 SeeBlue+2] [http://products.invitrogen.com/ivgn/product/LC5925 standard] into first and 5th lane<br />
**Mini-PROTEAN TGX Precast gels can only handle up to 30 μL of total solution<br />
**Mini-PROTEAN TGX Precast gels have 10 wells, plan accordingly<br />
*Load 25 μL sample into each gel well<br />
*record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/VgAuZ6dBOfs" frameborder="0" allowfullscreen></iframe></html><br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=971Western Blot Protocol2013-05-15T23:10:37Z<p>Monakhos: /* SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x [http://shop.expedeon.com/products/71-RunBlue-LDS-Antioxidant-and-Reducer/32-RunBlue-LDS-Sample-Buffer-10ml-4x-Conc/ RunBlue] LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/XnEdmk1Sqvg" frameborder="0" allowfullscreen></iframe></html><br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Prepare [[Mini-PROTEAN]] [http://www.bio-rad.com/prd/en/US/LSR/PDP/5cf78e19-7ed5-4373-a988-3e62456a488e/Mini-PROTEAN-Tetra-Cell Tetra Cell] tank for electrophoresis (see video)<br />
*Use these gels if doing electrophoresis on the Mini-PROTEAN rig: 4–20% Mini-PROTEAN TGX Precast Gel [http://www.bio-rad.com/prd/en/US/adirect/biorad?cmd=catProductDetail&vertical=LSR&country=US&lang=en&productID=456-1093 #456-1093]<br />
*Remove green tape strip from bottom of gel cassette<br />
*Set up gel cassette in Mini-PROTEAN gel holder, wells facing inward, and put in tank<br />
**If running only 1 or 2 gels remove the 2nd companion gel-holder<br />
*Remove comb from top of gel cassette<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment making sure RB covers wells <br />
*Fill exterior compartment with RB<br />
*Rinse wells with RB using pipette<br />
<br />
<br />
*Load [https://products.invitrogen.com/ivgn/product/LC5925 SeeBlue+2] [http://products.invitrogen.com/ivgn/product/LC5925 standard] into first and 5th lane<br />
**Mini-PROTEAN TGX Precast gels can only handle up to 30 μL of total solution<br />
**Mini-PROTEAN TGX Precast gels have 10 wells, plan accordingly<br />
*Load 25 μL sample into each gel well<br />
*record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/VgAuZ6dBOfs" frameborder="0" allowfullscreen></iframe></html><br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=970Western Blot Protocol2013-05-14T16:54:04Z<p>Monakhos: /* SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x [http://shop.expedeon.com/products/71-RunBlue-LDS-Antioxidant-and-Reducer/32-RunBlue-LDS-Sample-Buffer-10ml-4x-Conc/ RunBlue] LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/XnEdmk1Sqvg" frameborder="0" allowfullscreen></iframe></html><br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
<br />
*Load [https://products.invitrogen.com/ivgn/product/LC5925 SeeBlue+2] [http://products.invitrogen.com/ivgn/product/LC5925 standard] into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/VgAuZ6dBOfs" frameborder="0" allowfullscreen></iframe></html><br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=969Western Blot Protocol2013-05-14T16:51:05Z<p>Monakhos: /* SDS-PAGE STEP 1 -- PREP SAMPLE */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x [http://shop.expedeon.com/products/71-RunBlue-LDS-Antioxidant-and-Reducer/32-RunBlue-LDS-Sample-Buffer-10ml-4x-Conc/ RunBlue] LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/XnEdmk1Sqvg" frameborder="0" allowfullscreen></iframe></html><br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
<br />
*Load [https://products.invitrogen.com/ivgn/product/LC5925 SeeBlue+2] standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/VgAuZ6dBOfs" frameborder="0" allowfullscreen></iframe></html><br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=968Western Blot Protocol2013-05-14T16:24:18Z<p>Monakhos: /* 3. SDS-PAGE (GEL ELECTROPHORESIS) */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/XnEdmk1Sqvg" frameborder="0" allowfullscreen></iframe></html><br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
<br />
*Load [https://products.invitrogen.com/ivgn/product/LC5925 SeeBlue+2] standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/VgAuZ6dBOfs" frameborder="0" allowfullscreen></iframe></html><br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=967Western Blot Protocol2013-05-14T16:19:41Z<p>Monakhos: /* 4. BLOT TRANSFER (GEL TO MEMBRANE) */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/XnEdmk1Sqvg" frameborder="0" allowfullscreen></iframe></html><br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
<br />
*Load SeeBlue+2 standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/VgAuZ6dBOfs" frameborder="0" allowfullscreen></iframe></html><br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=966Western Blot Protocol2013-05-14T16:18:57Z<p>Monakhos: /* 3. SDS-PAGE (GEL ELECTROPHORESIS) */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<html><iframe width="560" height="315" src="http://www.youtube.com/embed/XnEdmk1Sqvg" frameborder="0" allowfullscreen></iframe></html><br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
<br />
*Load SeeBlue+2 standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=965Western Blot Protocol2013-05-14T16:14:50Z<p>Monakhos: /* 3. SDS-PAGE (GEL ELECTROPHORESIS) */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|500px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
<br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
<br />
*Load SeeBlue+2 standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=964Western Blot Protocol2013-05-14T16:12:56Z<p>Monakhos: /* SDS-PAGE STEP 1 -- PREP SAMPLE */</p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
<br />
[[File:SDS-PAGE.png|350px]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
*Load SeeBlue+2 standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
{{Clear}}<br />
{{Clear}}<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=963Western Blot Protocol2013-05-14T16:11:00Z<p>Monakhos: </p>
<hr />
<div><br />
{{Box|width=70%|WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN|<br />
;:::::::BRADLEY MONK<br />
;:::::::UCSD MOLECULAR COGNITION LAB<br />
}}<br />
<br />
{{Clear}}<br />
----<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
[[File:SDS-PAGE.png]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
*Load SeeBlue+2 standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
{{Clear}}<br />
{{Clear}}<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=962Western Blot Protocol2013-05-14T16:06:23Z<p>Monakhos: </p>
<hr />
<div>===WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN===<br />
<br />
===BRADLEY MONK===<br />
===UCSD MOLECULAR COGNITION LAB===<br />
<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
[[File:SDS-PAGE.png]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
{{Clear}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
*Load SeeBlue+2 standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
{{Clear}}<br />
<br />
{{Clear}}<br />
{{Clear}}<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
{{Clear}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
{{Box|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
{{Clear}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|width=300px|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
{{Clear}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__<br />
__NOEDITSECTION__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=961Western Blot Protocol2013-05-14T15:58:28Z<p>Monakhos: </p>
<hr />
<div>===WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN===<br />
<br />
===BRADLEY MONK===<br />
===UCSD MOLECULAR COGNITION LAB===<br />
<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
<br />
;Each sample will require a total of 500 μL HBR<br />
{{Box|float=right|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
[[File:SDS-PAGE.png]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|float=right|width=300px|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
*Load SeeBlue+2 standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|float=right|width=300px|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
<br />
{{Clear}}<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|float=right|width=300px|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png|300px]]<br />
}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|float=right|width=300px|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png|300px]]<br />
}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|width=300px|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
<br />
{{Box|float=right|width=300px|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|float=right|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Western_Blot_Protocol&diff=960Western Blot Protocol2013-05-14T15:49:23Z<p>Monakhos: Created page with "===WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN=== ===BRADLEY MONK=== ===UCSD MOLECULAR COGNITION LAB=== ==1. TISSUE COLLECTION== *Anesthetize rodent with is..."</p>
<hr />
<div>===WESTERN BLOT PROTOCOL FOR MEMBRANE BOUND RECEPTORS IN BRAIN===<br />
<br />
===BRADLEY MONK===<br />
===UCSD MOLECULAR COGNITION LAB===<br />
<br />
<br />
==1. TISSUE COLLECTION==<br />
*Anesthetize rodent with isofluorine (or equivalent) and decapitate<br />
*Extract [[brain]] and flash-freeze in 2-methylbutane cooled to -20oC with dry ice<br />
*Store [[brain]] at -80 in sucrose or OCT for no more than 1 week<br />
<br />
==2. TISSUE DISECTION & PREPARATION==<br />
*Prepare Homogenization Buffer Reagent (HBR) at 4 deg C<br />
*HBR: 0.32 M sucrose , 10 mM HEPES pH 7.4, 2 mM EDTA <br />
*Add protease & phosphatase inhibitor cocktail at 1:10 v/v into HBR<br />
*Each sample will require a total of 500 μL HBR<br />
{{Box|Why 500 μL ?|<br />
Each tissue section will be floated onto a 250 μL droplet of HBR directly on a glass slide (do not pre-float in a water bath). 100 μL of sample+HBR will be sucked off the slide with a pipette and dropped into a vial containing 200 μL HBR. Homogenize contents of vial and then centrifuge. The top 150 μL supernatant will be removed (this is the cytosol fraction - save if needed) leaving 150 μL HBR with the membrane pellet (precipitate). 50 μL HBR will be added to the membrane fraction bringing the volume back up to 200 μL. If collecting 6 samples per [[brain]], you will need to prep 3 mL HBR per [[brain]]<br />
}}<br />
<br />
*Keep stock HBR and aliquots chilled in ice bath as much as possible.<br />
*Aliquot 200 μL HBR (per section) into labeled vials. <br />
*Prepare a freeze-mount station for slides<br />
**this can be done simply by placing dry ice below a flat piece of glass or plastic<br />
*Place first slide onto freeze mount station to pre-cool<br />
*Use cryostat or freezing microtome to cut 200 μm frozen sections at -20oC<br />
**(remember to mark one side of [[brain]] with pin if hemisphere identification is important, or only cut one hemisphere at a time)<br />
*Collect target section on tip of soft-bristle brush<br />
*Pipette 250 μL droplet of HBR onto slide<br />
*Float tissue section onto this droplet and use brush to unfold<br />
*Once flat, use scalpel to dissect out region of interest (ROI)<br />
*Leave ROI on slide and remove unwanted tissue from slide <br />
*Use pipette to suck-up 100 μL of HBR+ROI tissue<br />
*Drop this 100 μL into the appropriate vial containing 200 μL HBR<br />
*Once all the ROI tissue sections are in vials, homogenize samples using a vortex or glass homogenizer<br />
*Use centrifuge to spin samples at 1000*g for 15 min <br />
*Using pipette, remove the top-most 150 μL from the sample (this is the cytosol fraction - save if needed) leaving the membrane fraction in 150 μL HBR<br />
*Add 50 μL more HBR to the membrane fraction vial bringing the total volume back up to 200 μL. <br />
*Use centrifuge to spin samples again at 1000*g for 60 min<br />
*Using pipette, remove the top-most 100 μL from the sample leaving the membrane fraction in a final volume of 100 μL HBR<br />
*Store sample at -80 deg C for no more than 1 week<br />
<br />
==3. SDS-PAGE (GEL ELECTROPHORESIS)==<br />
<br />
===SDS-PAGE STEP 1 -- PREP SAMPLE===<br />
[[File:SDS-PAGE.png]]<br />
<br />
*Thaw samples to 4oC and re-homogenize<br />
*Extract 30 μL from sample and drop in new vial<br />
*Add 10 μL 4x RunBlue LDS Loading Buffer with 5% DTT (LIFE sciences)<br />
*Heat at 95 deg C for 5 minutes<br />
*Vortex while hot and place on ice<br />
<br />
===SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS===<br />
[[File:Electrophoresis.png]]<br />
<br />
;Make Running Buffer (RB) <br />
{{Box|RUNNING BUFFER (RB)|<br />
;Make 1000 mL RB<br />
*950 mL diH2O<br />
*50 mL Tween-20<br />
}}<br />
<br />
*Remove white tape from gel plastic<br />
*Set up gel cassette in XCell mini-cell tank <br />
**use spacer if needed<br />
*Remove comb from top of gel plastic<br />
<br />
*Pour some RB into center gel compartment and look for leakage<br />
*Finish filling center gel compartment above white scaffold <br />
**make sure RB fills all the gel wells<br />
*Fill exterior compartment with RB<br />
**don’t overflow into center gel compartment<br />
<br />
*Load SeeBlue+2 standard into first lane<br />
*Load 40 μL sample into each gel well<br />
**record which lane contains which sample<br />
<br />
*Run gel 200V/50 minutes -- or until<br />
**dye is at the end of the plate<br />
**target band is in the center of the gel<br />
<br />
<br />
{{Box|Stopping Point|<br />
If you absolutely must store gel overnight, wrap gel in cellophane and keep at 4 deg C<br />
}}<br />
<br />
<br />
==4. BLOT TRANSFER (GEL TO MEMBRANE)==<br />
<br />
===BLOT TRANSFER STEP 1 -- PREP TRANSFER MATERIALS===<br />
<br />
;Make transfer buffer (TB)<br />
<br />
{{Box|TRANSFER BUFFER (TB)|<br />
;Make 1000 mL TB<br />
*750 mL diH2O<br />
*200 mL MeOH<br />
*50 mL 20x NuPAGE TB<br />
[[File:Transfer Buffer.png]]<br />
}}<br />
<br />
*Put gel in TB for 10 min<br />
<br />
*Put new membrane in 100% MeOH <br />
**leave in MeOH until turns opaque <br />
**usually less than < 20 sec<br />
*Remove membrane from MeOH and put in TB <br />
**leave in for 10 min<br />
<br />
===BLOT TRANSFER STEP 2 -- PREP CASSETTE===<br />
{{Box|Membrane Transfer Cassette|<br />
[[File:Membrane Cassette.png]]<br />
}}<br />
<br />
*Place cassette, dark side down and fill tray with TB<br />
*Roll out bubbles during each step<br />
**moisten everything going in cassette with TB<br />
**put a sponge in the cassette<br />
**put 2 Whattman papers on top of sponge<br />
**put the gel on top of Whattman papers<br />
**put the membrane on gel<br />
**put 2 Whattman papers on top membrane<br />
**put another sponge on top of Whattmans<br />
*Close frame with light side on top and lock.<br />
<br />
<br />
===BLOT TRANSFER STEP 3 -- RUN TRANSFER===<br />
<br />
*Add TB to Tetra Cell tank until 2/3 full<br />
*Place cassette in tank<br />
** lock should face upward<br />
** black side of cassette to black side of electrode insert <br />
*Fill tank to the fill-line with TB<br />
*Run 150V/1hr<br />
<br />
[[File:Blot Transfer.png]]<br />
<br />
<br />
==5. ANTIBODY EXPOSURE==<br />
<br />
===ANTIBODY EXPOSURE STEP 1 -- MAKE BLOCK BUFFER===<br />
<br />
;Make PBS-T Block Buffer (milk + PBS + Tween) <br />
*Use 5 g Carnation Powder Milk per 100 mL PBS-T<br />
**PBS<br />
**Tween20<br />
**Carnation powder milk<br />
<br />
{{Box|PBS-T-Block QUICK METHOD|<br />
;Make 1000 mL PBS-T<br />
*900 mL diH2O<br />
*100 mL PBS-10x<br />
*1 mL Tween-20<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
<br />
{{Box|PBS-T-Block DILUTION METHOD|<br />
:Make a 10% Tween solution<br />
*10 mL Tween20<br />
*90 mL PBS-1x<br />
<br />
;Make 1000 mL PBS-T<br />
*10 mL Tween-10%<br />
*950 mL PBS-1x<br />
<br />
;Make 100 mL PBS-T-Block <br />
*5 g Carnation <br />
*100 mL PBS-T<br />
}}<br />
<br />
===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===<br />
<br />
*Remove membrane from cassette <br />
*Wash membrane with PBS-T 2X<br />
*Place membrane in PBS-T-Block for 1hr.<br />
*Wash membrane 2X/5min with PBS-T<br />
<br />
[[File:Antibody Incubation.png]]<br />
<br />
===ANTIBODY EXPOSURE STEP 3 -- ANTIBODY INCUBATION===<br />
<br />
*Make 1° Antibody in PBS-T-Block. <br />
*Add membrane and incubate overnight at 4 deg C on rocker table. <br />
<br />
*Wash 4X/ 5min. with PBS-T.<br />
*Make 2° Antibody in PBS-T-Block. (Ms 1:200)<br />
*Add membrane and incubate 1 hr.<br />
*Wash 4X/5 min. with PBS-T. <br />
*Wash 3X/ 5 min. with diH2O.<br />
<br />
{{Box|Stopping Point|<br />
Incubation overnight should be done in a cold room at 4o C <br />
}}<br />
<br />
<br />
==6. BLOT VISUALIZATION==<br />
<br />
===BLOT VISUALIZATION STEP 1 -- PREP STATION===<br />
<br />
*Place saran wrap on top of upside down box tray<br />
*Have another box ready to place on top<br />
**this protects membrane and Dura from light<br />
*Place membrane on top of saran wrap<br />
<br />
<br />
===BLOT VISUALIZATION STEP 2 -- PREP DURA===<br />
<br />
*Pour Dura A in conical vial-A and Dura B in conical vial-B<br />
**use 4 mL total Dura per gel<br />
<br />
*Mix Dura A with Dura B and pour onto membrane<br />
**do this quickly <br />
**use drop method so Dura doesn’t run off membrane<br />
**protect from light by putting box on top <br />
*Incubate for 5 min<br />
*After the 5 min incubation, visualize blot in ECL machine ASAP!<br />
<br />
<br />
<br />
<br />
__NOTOC__</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=File:Antibody_Incubation.png&diff=959File:Antibody Incubation.png2013-05-14T15:28:18Z<p>Monakhos: </p>
<hr />
<div></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=File:Blot_Transfer.png&diff=958File:Blot Transfer.png2013-05-14T15:28:12Z<p>Monakhos: </p>
<hr />
<div></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=File:Membrane_Cassette.png&diff=957File:Membrane Cassette.png2013-05-14T15:27:53Z<p>Monakhos: </p>
<hr />
<div></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=File:Transfer_Buffer.png&diff=956File:Transfer Buffer.png2013-05-14T15:27:45Z<p>Monakhos: </p>
<hr />
<div></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=File:Electrophoresis.png&diff=955File:Electrophoresis.png2013-05-14T15:26:43Z<p>Monakhos: </p>
<hr />
<div></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=File:SDS-PAGE.png&diff=954File:SDS-PAGE.png2013-05-14T15:23:49Z<p>Monakhos: Monakhos uploaded a new version of &quot;File:SDS-PAGE.png&quot;</p>
<hr />
<div></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=File:SDS-PAGE.png&diff=953File:SDS-PAGE.png2013-05-14T15:23:47Z<p>Monakhos: Monakhos uploaded a new version of &quot;File:SDS-PAGE.png&quot;</p>
<hr />
<div></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=File:SDS-PAGE.png&diff=952File:SDS-PAGE.png2013-05-14T15:23:32Z<p>Monakhos: </p>
<hr />
<div></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Brain_Informatics&diff=951Brain Informatics2013-05-14T03:21:07Z<p>Monakhos: </p>
<hr />
<div><html><iframe width="420" height="315" src="http://www.youtube.com/embed/KJJdXtmA4pQ" frameborder="0" allowfullscreen></iframe></html><br />
<br />
<html><iframe src="http://www.neuinfo.org/downloads/nif_navigator_1.2/nif_navigator.html" height="775" width="250" frameborder="0" seamless="seamless" style="float:right"></iframe><br />
</html><br />
<br />
{| class="wikitable" style="background-color:white;"<br />
|+Neural Informatics Tools<br />
|-<br />
! Link !! Description<br />
|-<br />
|[http://api.brain-map.org/examples/svgoverlay/index.html Allen Brain Atlas API] || Allen Institute's annotated reference atlases have SVG files that contain structural boundaries.<br />
|-<br />
|[http://www.nrcam.uchc.edu/vcell_software/user_guide.html VCell] || Most useful cell modeling tool I've seen so far.<br />
|-<br />
|[http://vcell.org/bionetgen/ BioNetGen] || A web-based tool for automatically generating a biochemical reaction networks from user-specified rules for biomolecular interactions. <br />
|-<br />
|[http://bionumbers.hms.harvard.edu/ B10NUMB3R5] || (BIONUMBERS) A database of useful biological numbers. <br />
|-<br />
|[http://www.bioinformaticsservices.com/ BioInformatics Services] || BioInformatics Services is a biotechnology consulting firm specializing in using computational biology to solve complex biological and data analysis problems. <br />
|-<br />
|[http://www.cellmigration.org/resource/modeling/model_resources.shtml#softwares cellmigrationgateway] || modeling software <br />
|-<br />
|[http://www.cellml.org/ CellML] || The CellML language is an open standard based on the XML markup language at the University of Auckland and affiliated research groups. <br />
|-<br />
|[https://sites.google.com/site/compcellbio/ CompCell Bio Web] || Development site for teaching modules on quantitative cell biology. <br />
|-<br />
|[http://doqcs.ncbs.res.in/ DOQCS] || Quantitative Cellular Signaling (DOQCS) is a repository of models of signaling pathways. <br />
|-<br />
|[http://www.e-cell.org/ecell/ E-Cell] || A Multi-Algorithm, Multi-Timescale Simulation Software Environment. <br />
|-<br />
|[http://gfit.sourceforge.net/ Gfit] || A program for global regression analysis of experimental data. <br />
|-<br />
|[http://www.kitware.com/ Kitware] || Professional Visualization Solutions, Tools and Support. <br />
|-<br />
|[http://www.mcell.cnl.salk.edu/ MCell] || General Monte Carlo Simulator of Cellular Microphysiology. <br />
|-<br />
|[http://mnm.engr.uconn.edu/MNM/SMSSearchServlet Minimotif Miner] || Application to search for short sequence motifs in Proteins. <br />
|}<br />
<br />
{{Box|width=255px|Bioinformatics Tools|<br />
*[http://bbid.grc.nia.nih.gov/ BBID-Biological Biochemical Image Database]<br />
}}</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Brain_Informatics&diff=950Brain Informatics2013-05-14T03:19:27Z<p>Monakhos: </p>
<hr />
<div><br />
<br />
<html><iframe src="http://www.neuinfo.org/downloads/nif_navigator_1.2/nif_navigator.html" height="775" width="250" frameborder="0" seamless="seamless" style="float:right"></iframe><br />
</html><br />
<br />
{| class="wikitable" style="background-color:white;"<br />
|+Neural Informatics Tools<br />
|-<br />
! Link !! Description<br />
|-<br />
|[http://api.brain-map.org/examples/svgoverlay/index.html Allen Brain Atlas API] || Allen Institute's annotated reference atlases have SVG files that contain structural boundaries.<br />
|-<br />
|[http://www.nrcam.uchc.edu/vcell_software/user_guide.html VCell] || Most useful cell modeling tool I've seen so far.<br />
|-<br />
|[http://vcell.org/bionetgen/ BioNetGen] || A web-based tool for automatically generating a biochemical reaction networks from user-specified rules for biomolecular interactions. <br />
|-<br />
|[http://bionumbers.hms.harvard.edu/ B10NUMB3R5] || (BIONUMBERS) A database of useful biological numbers. <br />
|-<br />
|[http://www.bioinformaticsservices.com/ BioInformatics Services] || BioInformatics Services is a biotechnology consulting firm specializing in using computational biology to solve complex biological and data analysis problems. <br />
|-<br />
|[http://www.cellmigration.org/resource/modeling/model_resources.shtml#softwares cellmigrationgateway] || modeling software <br />
|-<br />
|[http://www.cellml.org/ CellML] || The CellML language is an open standard based on the XML markup language at the University of Auckland and affiliated research groups. <br />
|-<br />
|[https://sites.google.com/site/compcellbio/ CompCell Bio Web] || Development site for teaching modules on quantitative cell biology. <br />
|-<br />
|[http://doqcs.ncbs.res.in/ DOQCS] || Quantitative Cellular Signaling (DOQCS) is a repository of models of signaling pathways. <br />
|-<br />
|[http://www.e-cell.org/ecell/ E-Cell] || A Multi-Algorithm, Multi-Timescale Simulation Software Environment. <br />
|-<br />
|[http://gfit.sourceforge.net/ Gfit] || A program for global regression analysis of experimental data. <br />
|-<br />
|[http://www.kitware.com/ Kitware] || Professional Visualization Solutions, Tools and Support. <br />
|-<br />
|[http://www.mcell.cnl.salk.edu/ MCell] || General Monte Carlo Simulator of Cellular Microphysiology. <br />
|-<br />
|[http://mnm.engr.uconn.edu/MNM/SMSSearchServlet Minimotif Miner] || Application to search for short sequence motifs in Proteins. <br />
|}<br />
<br />
{{Box|width=255px|Bioinformatics Tools|<br />
*[http://bbid.grc.nia.nih.gov/ BBID-Biological Biochemical Image Database]<br />
}}</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Brain_Informatics&diff=949Brain Informatics2013-05-14T03:16:50Z<p>Monakhos: Created page with "[http://api.brain-map.org/examples/svgoverlay/index.html Allen Brain Atlas API]"</p>
<hr />
<div>[http://api.brain-map.org/examples/svgoverlay/index.html Allen Brain Atlas API]</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=VCell&diff=948VCell2013-05-14T02:47:35Z<p>Monakhos: </p>
<hr />
<div><div class="wpImageAnnotatorEnable"><br />
<span class="wpImageAnnotatorPageName" style="display:none;">[[VCell]]</span><br />
<span class="wpImageAnnotatorFullName" style="display:none;">[[VCell]]</span><br />
<div class="wpImageAnnotatorFile">[[File:VCell.jpg]]</div><br />
<div style="display:none;"><div><div><!--Dummy marker to have image notes inserted below this line-->{{ImageNoteEnd|id=-1}}<br />
</div><br />
</div><br />
<br />
<br />
<br />
<nowiki><br />
{{subst:ImageWithNotes|img=''[[File:VCell.jpg]]''}}<br />
</nowiki><br />
<br />
[[File:Qdot core shell.png|200px]]<br />
<br />
[[File:Qdot core shell.png|thumb|200px]]<br />
<br />
<br />
<br />
[http://www.nrcam.uchc.edu/vcell_software/user_guide.html?current=four VCell Tutorial]</div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:X&diff=947Template:X2013-05-14T02:37:17Z<p>Monakhos: Created page with "{{{{{|safesubst:}}}#switch:{{{1}}} <!-- __behaviour switches__ --> | NOTOC | FORCETOC | TOC | NOEDITSECTION | NEWSECTIONLINK | NONEWSECTIONLINK | NOCONTENTCONVERT | NOCC |..."</p>
<hr />
<div>{{{{{|safesubst:}}}#switch:{{{1}}}<br />
<!-- __behaviour switches__ --><br />
| NOTOC | FORCETOC | TOC<br />
| NOEDITSECTION | NEWSECTIONLINK | NONEWSECTIONLINK<br />
| NOCONTENTCONVERT | NOCC<br />
| NOTITLECONVERT | NOTC<br />
| NOGALLERY<br />
| END | START <br />
| NOINDEX | INDEX<br />
| STATICREDIRECT<br />
| HIDDENCAT<br />
= &#95;_[[mw:Help:Magic_words#Behavior_switches|{{{2|{{{1}}}}}}]]_&#95;<br />
<br />
<!-- {{behavior switches:}} --><br />
| PAGESIZE<br />
| PROTECTIONLEVEL<br />
| DISPLAYTITLE<br />
| DEFAULTSORT | DEFAULTSORTKEY | DEFAULTCATEGORYSORTKEY<br />
| PAGESINCATEGORY | PAGESINCAT<br />
| NUMBERINGROUP | NUMINGROUP<br />
| PAGESINNAMESPACE | PAGESINNS<br />
= &#123;&#123;[[mw:Help:Magic_words|{{{2|{{{1}}}:}}}]]&#125;&#125;<br />
<br />
<!-- #redirect: --><br />
| REDIRECT | redirect<br />
= [[mw:Help:Redirects|{{{2|&#35;{{{1}}}}}}]]<br />
<br />
<!-- {{parser functions:}} --><br />
| localurl | localurle | fullurl | fullurle | filepath<br />
| urlencode | anchorencode<br />
= &#123;&#123;[[mw:Help:Magic_words#URL_data|{{{2|{{{1}}}:}}}]]&#125;&#125;<br />
| ns | nse<br />
= &#123;&#123;[[mw:Help:Magic_words#Namespaces|{{{2|{{{1}}}:}}}]]&#125;&#125;<br />
| lc | lcfirst | uc | ucfirst<br />
| formatnum<br />
| padleft | padright<br />
| grammar | plural<br />
= &#123;&#123;[[mw:Help:Magic_words#Formatting|{{{2|{{{1}}}:}}}]]&#125;&#125;<br />
| GENDER | gender<br />
| int | msg | msgnw | raw<br />
= &#123;&#123;[[mw:Help:Magic_words#Miscellaneous|{{{2|{{{1}}}:}}}]]&#125;&#125;<br />
| subst | safesubst<br />
= &#123;&#123;[[m:Help:Substitution|{{{2|{{{1}}}:}}}]]&#125;&#125;<br />
<br />
<!-- {{#parser functions:}} --><br />
| formatdate | dateformat<br />
= &#123;&#123;[[mw:Help:Magic_words#Formatting|{{{2|&#35;{{{1}}}:}}}]]&#125;&#125;<br />
| special<br />
| tag<br />
| language<br />
= &#123;&#123;[[mw:Help:Magic_words#Miscellaneous|{{{2|&#35;{{{1}}}:}}}]]&#125;&#125;<br />
<br />
<!-- {{#ParserFunctions:}} --><br />
| expr | if | ifeq | ifexist | ifexpr | iferror<br />
| rel2abs | switch | time | timel | titleparts<br />
= &#123;&#123;[[mw:Help:Extension:ParserFunctions#.23{{{1}}}:|{{{2|&#35;{{{1}}}:}}}]]&#125;&#125;<br />
<br />
<!-- {{variables}} --><br />
| SITENAME | SERVER | SERVERNAME | SCRIPTPATH<br />
| DIRMARK | DIRECTIONMARK<br />
| CURRENTVERSION<br />
| CONTENTLANGUAGE | CONTENTLANG<br />
| REVISIONID | REVISIONUSER<br />
| REVISIONDAY | REVISIONDAY2 | REVISIONMONTH | REVISIONYEAR | REVISIONTIMESTAMP<br />
= &#123;&#123;[[mw:Help:Magic_words#Technical_metadata|{{{2|{{{1}}}}}}]]&#125;&#125;<br />
| NUMBEROFPAGES | NUMBEROFARTICLES | NUMBEROFFILES | NUMBEROFEDITS | NUMBEROFVIEWS<br />
| NUMBEROFUSERS | NUMBEROFADMINS | NUMBEROFACTIVEUSERS<br />
= &#123;&#123;[[mw:Help:Magic_words#Statistics|{{{2|{{{1}}}}}}]]&#125;&#125;<br />
| NAMESPACE | NAMESPACEE | SUBJECTSPACE | SUBJECTSPACEE<br />
| ARTICLESPACE | ARTICLESPACEE | TALKSPACE | TALKSPACEE<br />
= &#123;&#123;[[mw:Help:Magic_words#Namespaces|{{{2|{{{1}}}}}}]]&#125;&#125;<br />
| CURRENTMONTHNAMEGEN | LOCALMONTHNAMEGEN<br />
= &#123;&#123;[[m:Help:Magic_words#Language-dependent_word_conversions|{{{2|{{{1}}}}}}]]&#125;&#125;<br />
<br />
<!-- <extension tags> --><br />
| categorytree<br />
= <[[mw:Extension:CategoryTree|{{{2|{{{1}}}}}}]]><br />
| charinsert<br />
= <[[mw:Extension:CharInsert|{{{2|{{{1}}}}}}]]><br />
| hiero<br />
= <[[mw:Extension:WikiHiero|{{{2|{{{1}}}}}}]]><br />
| imagemap<br />
= <[[mw:Extension:ImageMap|{{{2|{{{1}}}}}}]]><br />
| inputbox<br />
= <[[mw:Extension:InputBox|{{{2|{{{1}}}}}}]]><br />
| poem<br />
= <[[mw:Extension:Poem|{{{2|{{{1}}}}}}]]><br />
| ref | references<br />
= <[[mw:Extension:Cite/Cite.php|{{{2|{{{1}}}}}}]]><br />
| source | syntaxhighlight<br />
= <[[mw:Extension:SyntaxHighlight GeSHi|{{{2|{{{1}}}}}}]]><br />
| timeline<br />
= <[[mw:Extension:EasyTimeline|{{{2|{{{1}}}}}}]]><br />
<br />
<!-- other {{variables}} / anything else --><br />
| &#123;&#123;[[mw:Help:Magic words|{{{2|{{{1}}}}}}]]&#125;&#125;<br />
}}<noinclude><br />
<br />
<br />
----<br />
Links to a [[help]] page about the magic word and automatically adds the correct syntax (case sensitive in most cases).<br />
<br />
* <nowiki>{{</nowiki>x|NAMESPACE}} → {{x|NAMESPACE}}<br />
* <nowiki>{{</nowiki>x|DEFAULTSORT}} → {{x|DEFAULTSORT}}<br />
* <nowiki>{{</nowiki>x|ifexist}} → {{x|ifexist}}<br />
* <nowiki>{{</nowiki>x|localurl}} → {{x|localurl}}<br />
* <nowiki>{{</nowiki>x|REDIRECT}} → {{x|REDIRECT}}<br />
* <nowiki>{{</nowiki>x|safesubst}} → {{x|safesubst}}<br />
* <nowiki>{{</nowiki>x|NOTOC}} → {{x|NOTOC}}<br />
* <nowiki>{{</nowiki>x|categorytree}} → {{x|categorytree}}<br />
...<br />
<br />
[[Category:Interwiki link templates|{{PAGENAME}}]]<br />
[[Category:Typing-aid templates|{{PAGENAME}}]]<br />
</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:Tlc&diff=946Template:Tlc2013-05-14T02:36:06Z<p>Monakhos: Created page with "<span style="white-space:nowrap;"><code>&#123;&#123;{{#if:{{{1|}}}|{{{1}}}| tlc&#124;...}}<!-- -->{{#ifeq:{{{2|x}}}|{{{2|}}}| &#124;{{{2}}} | }}<!-- -->{{#ifeq:{{{3|x}}}|{{{3|..."</p>
<hr />
<div><span style="white-space:nowrap;"><code>&#123;&#123;{{#if:{{{1|}}}|{{{1}}}| tlc&#124;...}}<!--<br />
-->{{#ifeq:{{{2|x}}}|{{{2|}}}| &#124;{{{2}}} | }}<!--<br />
-->{{#ifeq:{{{3|x}}}|{{{3|}}}| &#124;{{{3}}} | }}<!--<br />
-->{{#ifeq:{{{4|x}}}|{{{4|}}}| &#124;{{{4}}} | }}<!--<br />
-->{{#ifeq:{{{5|x}}}|{{{5|}}}| &#124;{{{5}}} | }}<!--<br />
-->{{#ifeq:{{{6|x}}}|{{{6|}}}| &#124;{{{6}}} | }}<!--<br />
-->{{#ifeq:{{{7|x}}}|{{{7|}}}| &#124;{{{7}}} | }}<!--<br />
-->{{#ifeq:{{{8|x}}}|{{{8|}}}| &#124;{{{8}}} | }}<!--<br />
-->{{#ifeq:{{{9|x}}}|{{{9|}}}| &#124;{{{9}}} | }}<!--<br />
-->&#125;&#125;</code></span><noinclude><br />
{{documentation}}<br />
</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:NW2&diff=945Template:NW22013-05-14T02:35:26Z<p>Monakhos: Created page with "<span style="white-space:nowrap">{{#switch:{{{2}}} |c|tt={{#ifeq:{{{2}}}|c|<code>|<tt>}}{{#tag:nowiki|{{{3}}}}}{{#ifeq:{{{2}}}|c|</code>|</tt>}} |nw={{#ifeq:{{{1}}}|c|<code>..."</p>
<hr />
<div><span style="white-space:nowrap">{{#switch:{{{2}}}<br />
|c|tt={{#ifeq:{{{2}}}|c|<code>|<tt>}}{{#tag:nowiki|{{{3}}}}}{{#ifeq:{{{2}}}|c|</code>|</tt>}}<br />
|nw={{#ifeq:{{{1}}}|c|<code>|<tt>}}{{#tag:nowiki|{{{3}}}}}{{#ifeq:{{{1}}}|c|</code>|</tt>}}<br />
|{{#switch:{{{1}}}<br />
|c=<code>{{{2}}}</code><br />
|tt=<tt>{{{2}}}</tt><br />
|nw={{#tag:nowiki|{{{2}}}}}<br />
|{{{1}}}<br />
}}<br />
}}</span><noinclude><br />
<br />
<br />
----<br />
Prevents wrapping of lines like {{tl|nowrap}} (redirect: {{tl|NW}}) plus the option to also apply &lt;tt>/&lt;code> tags or &lt;nowiki> tags or both. This also works without any of those but if all you want is the no wrapping part use {{tl|NW}} instead.<br />
<br />
Note: The nowiki option makes use of {{x|tag}}, which doesn't work correctly with templates.<br />
<br />
The order doesn't matter as long as the ''text'' goes last (e.g. {{tlf|NW2|c|nw|''text''}} is the same as {{tlf|NW2|nw|c|''text''}}).<br />
* {{tlc|NW2|c|''text''}} - nowrap + &lt;code><br />
* {{tlc|NW2|tt|''text''}} - nowrap + &lt;tt><br />
* {{tlc|NW2|nw|''text''}} - nowrap + &lt;nowiki><br />
* {{tlc|NW2|c|nw|''text''}} - nowrap + &lt;code> + &lt;nowiki><br />
* {{tlc|NW2|nw|tt|''text''}} - nowrap + &lt;tt> + &lt;nowiki><br />
<br />
[[Category:Formatting templates|{{PAGENAME}}]]<br />
</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:Translated_tag&diff=944Template:Translated tag2013-05-14T02:33:35Z<p>Monakhos: Created page with "{{#ifeq:{{NAMESPACE}}|{{ns:4}}||<!-- ignore commons namespace for demonstration pages --> {{#switch:{{FULLPAGENAME}} |{{{ns|{{{ns1|{{ns:10}}}}}}}}:{{ucfirst:{{{page|{{{page1..."</p>
<hr />
<div>{{#ifeq:{{NAMESPACE}}|{{ns:4}}||<!-- ignore commons namespace for demonstration pages --><br />
{{#switch:{{FULLPAGENAME}} <br />
|{{{ns|{{{ns1|{{ns:10}}}}}}}}:{{ucfirst:{{{page|{{{page1}}}}}}}}{{{sub|/{{SUBPAGENAME}}}}}<br />
|{{{ns2|{{ns:10}}}}}:{{ucfirst:{{{page2}}}}}{{{sub|/{{SUBPAGENAME}}}}}<br />
|{{{ns3|{{ns:10}}}}}:{{ucfirst:{{{page3}}}}}{{{sub|/{{SUBPAGENAME}}}}}<br />
|{{{ns4|{{ns:10}}}}}:{{ucfirst:{{{page4}}}}}{{{sub|/{{SUBPAGENAME}}}}} = <!-- for any exceptions --><br />
|#default =<br />
{{#switch:localisation|{{{1}}}|{{{2}}}=<!-- it may be used directly so the message doesn't apply -->|#default =<br />
<div class="translatedTag" style="text-align:center"><br />
{{#ifexist:Template:Translated tag/{{{lang|{{SUBPAGENAME}}}}}<!-- check if we have a translation --><br />
| {{Translated tag/{{{lang|{{SUBPAGENAME}}}}}|{{{template|{{#ifexist:{{BASEPAGENAME}}|{{BASEPAGENAME}}|{{#titleparts:{{PAGENAME}}|1}}}}}}}}}<br />
}}<br />
<div>{{lang|en|'''''NOTE:'' Please do ''not'' use this template directly! This is just for translation.<!--<br />
-->{{#ifeq:{{NAMESPACE}}|{{ns:10}}<!-- leave this part out on non-template pages because it won't make sense --><br />
| &#32;Use {{tl|{{{template|{{#ifexist:{{BASEPAGENAME}}|{{BASEPAGENAME}}|{{#titleparts:{{PAGENAME}}|1}}}}}}}}} instead!'''<br />
}}<!--<br />
-->}}</div><br />
</div><br />
}}<!--<br />
auto-categorizer<br />
-->{{#ifeq:{{FULLPAGENAME}}|{{ns:10}}:{{BASEPAGENAME}}/{{SUBPAGENAME}}<br />
| {{DEFAULTSORT:{{PAGENAME}}}}<!--<br />
-->{{{localcat|[[Category:{{#ifexist:Template:Translated tag/local category-{{SUBPAGENAME}}<br />
| {{translated tag/local category-{{SUBPAGENAME}}|{{{1}}}}}<br />
| Commons templates-{{SUBPAGENAME}} }}]]}}}<!--<br />
-->[[Category:Translated {{#switch:{{lc:{{{1}}}}}<br />
| cleanup = media cleanup templates<br />
| license = license tags<br />
| marker = marker templates<br />
| problem = problem tags<br />
| restriction = restriction tags<br />
| header = header templates<br />
| infobox = infobox templates<br />
| navigational = navigational templates<br />
| protection = protection templates<br />
| source = source templates<br />
| documentation = documentation templates<br />
| user = user templates<br />
| localisation = templates for localisation<br />
| templates needing categories<br />
}}]]<br />
| {{#ifeq:{{NAMESPACE}}|{{ns:6}}|[[Category:Images using translated tags]]}}<!-- check for misuse --><br />
}}<!--<br />
end auto-categorizer<br />
-->}}<br />
}}<noinclude>{{documentation}}</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:Must_be_substituted/layout&diff=943Template:Must be substituted/layout2013-05-14T02:32:45Z<p>Monakhos: Created page with "{| class="messagebox vcard layouttemplate" style="direction: {{Dir|{{{lang|}}}}}" |style="width:37px"|Notice |{{{text|empty}}} ---- {{Mus..."</p>
<hr />
<div>{| class="messagebox vcard layouttemplate" style="direction: {{Dir|{{{lang|}}}}}"<br />
|style="width:37px"|[[File:Info non-talk.svg|35px|link=|Notice]]<br />
|{{{text|empty}}}<br />
----<br />
{{Must be substituted/lang}}<br />
|}<noinclude><br />
<br />
[[Category:Layout templates|{{PAGENAME}}]]<br />
</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:Must_be_substituted/en&diff=942Template:Must be substituted/en2013-05-14T02:31:58Z<p>Monakhos: Created page with "{{Must be substituted/layout |text=<big>Please note that this template '''must be substituted'''. Replace {{NW2|tt|{&#123;{{{name|{{lcfirst:{{PAGENAME}..."</p>
<hr />
<div>{{Must be substituted/layout<br />
|text=<big>Please note that this template '''must be [[m:help:substitution|substituted]]'''. Replace {{NW2|tt|{&#123;{{{name|{{lcfirst:{{PAGENAME}}}}}}}&#125;&#125;}} with {{NW2|tt|{&#123;subst:{{{name|{{lcfirst:{{PAGENAME}}}}}}}&#125;&#125;}}</big>.<br /><small><span class="plainlinks">[{{fullurl:Special:WhatLinksHere/{{FULLPAGENAMEE}}|hideredirs=1&hidelinks=1&hidetrans=0}} Check un-substituted usage]</span><br />
|lang=en<br />
}}<noinclude><br />
{{translated tag|marker}}<br />
</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:Fallback&diff=941Template:Fallback2013-05-14T02:31:02Z<p>Monakhos: Created page with "{{#ifexist: Template:{{{1|}}}/{{#if: {{{2|}}} |{{{2}}}|{{int:Lang}}}} | {{#if: {{{2|}}} |{{{2}}}|{{int:Lang}}}} | {{#ifexist: Template:{{{1|}}}/{{GetFallback|{{#if: {{{2|}}} |..."</p>
<hr />
<div>{{#ifexist: Template:{{{1|}}}/{{#if: {{{2|}}} |{{{2}}}|{{int:Lang}}}} | {{#if: {{{2|}}} |{{{2}}}|{{int:Lang}}}} | {{#ifexist: Template:{{{1|}}}/{{GetFallback|{{#if: {{{2|}}} |{{{2}}}|{{int:Lang}}}}}} | {{GetFallback|{{#if: {{{2|}}} |{{{2}}}|{{int:Lang}}}}}} | {{#ifexist: Template:{{{1|}}}/{{GetFallback2|{{#if: {{{2|}}} |{{{2}}}|{{int:Lang}}}}}} | {{GetFallback2|{{#if: {{{2|}}} |{{{2}}}|{{int:Lang}}}}}} | {{CONTENTLANGUAGE}} }} }} }}<noinclude><br />
<br />
{{documentation}}<br />
[[Category:Localisation templates]]<br />
</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:Autotranslate&diff=940Template:Autotranslate2013-05-14T02:30:06Z<p>Monakhos: Created page with "<includeonly>{{{{{base|}}}/{{Fallback|{{{base|}}}|{{{lang|{{int:Lang}} }}} }} | 1={{{ 1|}}}| 2={{{ 2|}}}| 3={{{ 3|}}}| 4={{{ 4|}}}| 5={{{ 5|}}} | 6={{{ 6|}}}| 7={{{ 7|}}}| 8={..."</p>
<hr />
<div><includeonly>{{{{{base|}}}/{{Fallback|{{{base|}}}|{{{lang|{{int:Lang}} }}} }}<br />
| 1={{{ 1|}}}| 2={{{ 2|}}}| 3={{{ 3|}}}| 4={{{ 4|}}}| 5={{{ 5|}}}<br />
| 6={{{ 6|}}}| 7={{{ 7|}}}| 8={{{ 8|}}}| 9={{{ 9|}}}|10={{{10|}}}<br />
|11={{{11|}}}|12={{{12|}}}|13={{{13|}}}|14={{{14|}}}|15={{{15|}}} }}<!-- <br />
-->{{#ifeq: {{FULLPAGENAME}} | Template:{{{base|}}} | [[Category:Autotranslated templates|{{PAGENAME}}]]}}</includeonly><noinclude><br />
<br />
{{documentation}}<br />
<br />
[[Category:Autotranslated templates| *]]<br />
<br />
</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=Template:Must_be_substituted&diff=939Template:Must be substituted2013-05-14T02:29:28Z<p>Monakhos: Created page with "{{autotranslate|base=Must be substituted}} <includeonly> {{PAGENAME}} </includeonly><noinclude> {{documentation}} [[Category:T..."</p>
<hr />
<div>{{autotranslate|base=Must be substituted}}<br />
<includeonly><br />
[[Category:Templates which must be substituted|{{PAGENAME}}]]<br />
</includeonly><noinclude><br />
{{documentation}}<br />
[[Category:Template namespace templates|{{PAGENAME}}]]<br />
[[Category:Marker templates|{{PAGENAME}}]]<br />
</noinclude></div>Monakhoshttps://bradleymonk.com/wiki/index.php?title=VCell&diff=938VCell2013-05-14T02:27:33Z<p>Monakhos: </p>
<hr />
<div><div class="wpImageAnnotatorEnable"><br />
<span class="wpImageAnnotatorPageName" style="display:none;">[[VCell]]</span><br />
<span class="wpImageAnnotatorFullName" style="display:none;">[[VCell]]</span><br />
<div class="wpImageAnnotatorFile">[[File:VCell.jpg]]</div><br />
<div style="display:none;"><div><div><!--Dummy marker to have image notes inserted below this line-->{{ImageNoteEnd|id=-1}}<br />
</div><br />
</div><br />
<br />
<br />
{{subst:void|<span class="error">'''You forgot to subst this template. Replace &#123;{ImageWithNotes}&#125; with &#123;{subst:ImageWithNotes}&#125;'''</span>}}<div class="wpImageAnnotatorEnable"><br />
<span class="wpImageAnnotatorPageName" style="display:none;">VCell</span><br />
<span class="wpImageAnnotatorFullName" style="display:none;">VCell</span><br />
<div class="wpImageAnnotatorFile">''[[File:VCell.jpg]]''</div><br />
<div style="display:none;"><div><div><!--Dummy marker to have image notes inserted below this line-->{{ImageNoteEnd|id=-1}}<br />
</div><br />
</div><br />
<br />
<nowiki><br />
{{subst:ImageWithNotes|img=''[[File:VCell.jpg]]''}}<br />
</nowiki><br />
<br />
[[File:Qdot core shell.png|200px]]<br />
<br />
[[File:Qdot core shell.png|thumb|200px]]<br />
<br />
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[http://www.nrcam.uchc.edu/vcell_software/user_guide.html?current=four VCell Tutorial]</div>Monakhos