Western Blot Protocol - Revision history

Bradley Monk: /* ANTIBODY INCUBATION */

2013-05-23T22:48:45Z

ANTIBODY INCUBATION

← Older revision		Revision as of 15:48, 23 May 2013
Line 223:		Line 223:
	\|		\|
	*Dilute 1° Antibody in PBSTb		*Dilute 1° Antibody in PBSTb
	** seal ~~each~~ membrane with 5 mL of PBSTb + 1°		** seal membrane with 8 mL of PBSTb + 1°
	**dilute 1° antibody 1000:1 in PBSTb		**dilute 1° antibody 1000:1 in PBSTb
	**aka dilute 5 uL 1° in 5 mL PBSTb		**aka dilute 8 uL 1° in 8 mL PBSTb
	*Put membrane in plastic sealer bag		*Put membrane in plastic sealer bag
	*Seal 3 sides of plastic		*Seal 3 sides of plastic
Line 247:		Line 247:
	*Dilute 2° Antibody in PBSTb		*Dilute 2° Antibody in PBSTb
	**Ms 1:200		**Ms 1:200
	** seal ~~each~~ membrane with 5 mL of PBSTb + 2°		** seal membrane with 8 mL of PBSTb + 2°
	**dilute 2° antibody 1000:1 in PBSTb		**dilute 2° antibody 1000:1 in PBSTb
	**aka dilute 5 uL 2° in 5 mL PBSTb		**aka dilute 8 uL 2° in 8 mL PBSTb
	*Put membrane in plastic sealer bag		*Put membrane in plastic sealer bag
	*Seal 3 sides of plastic		*Seal 3 sides of plastic

Bradley Monk: /* ANTIBODY INCUBATION */

2013-05-23T22:28:55Z

ANTIBODY INCUBATION

← Older revision		Revision as of 15:28, 23 May 2013
Line 223:		Line 223:
	\|		\|
	*Dilute 1° Antibody in PBSTb		*Dilute 1° Antibody in PBSTb
			** seal each membrane with 5 mL of PBSTb + 1°
			**dilute 1° antibody 1000:1 in PBSTb
			**aka dilute 5 uL 1° in 5 mL PBSTb
	*Put membrane in plastic sealer bag		*Put membrane in plastic sealer bag
	*Seal 3 sides of plastic		*Seal 3 sides of plastic
Line 244:		Line 247:
	*Dilute 2° Antibody in PBSTb		*Dilute 2° Antibody in PBSTb
	**Ms 1:200		**Ms 1:200
			** seal each membrane with 5 mL of PBSTb + 2°
			**dilute 2° antibody 1000:1 in PBSTb
			**aka dilute 5 uL 2° in 5 mL PBSTb
	*Put membrane in plastic sealer bag		*Put membrane in plastic sealer bag
	*Seal 3 sides of plastic		*Seal 3 sides of plastic
	*Pour in 1° Antibody PBSTb mixture		*Pour in 2° Antibody PBSTb mixture
	*Incubate for 1 h at 4° C on rocker table.		*Incubate for 1 h at 4° C on rocker table.
	\|		\|

Bradley Monk at 02:40, 22 May 2013

2013-05-22T02:40:40Z

← Older revision		Revision as of 19:40, 21 May 2013
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	*put icepack inside tank, and put tank in ice bath		*put icepack inside tank, and put tank in ice bath
	*Fill tank to the fill-line with TB		*Fill tank to the fill-line with TB
	*Run ~~100V~~/1hr		*Run 150V/1hr

	;After transfer, identify sample positions on membrane		;After transfer, identify sample positions on membrane

Bradley Monk: /* SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS */

2013-05-21T23:50:14Z

SDS-PAGE STEP 2 -- RUN GEL ELECTROPHORESIS

← Older revision		Revision as of 16:50, 21 May 2013
Line 99:		Line 99:


	*Run gel ~~100V~~ until marker dye runs off bottom of gel		*Run gel 200V until marker dye runs off bottom of gel
	**approx. 30 min		**approx. 30 min
	**ideally run until target band is in center of gel		**ideally run until target band is in center of gel

Bradley Monk at 01:20, 19 May 2013

2013-05-19T01:20:49Z

← Older revision		Revision as of 18:20, 18 May 2013
Line 219:		Line 219:
	=== ANTIBODY INCUBATION ===		=== ANTIBODY INCUBATION ===

			{{2Box\|width1=300\|width2=300\|
	;*Prepare for 1° Antibody Incubation		Prepare for 1° Antibody Incubation
			\|
	*Dilute 1° Antibody in PBSTb		*Dilute 1° Antibody in PBSTb
	*Put membrane in plastic sealer bag		*Put membrane in plastic sealer bag
Line 226:		Line 227:
	*Pour in 1° Antibody PBSTb mixture		*Pour in 1° Antibody PBSTb mixture
	*Incubate for 24 h at 4° C on rocker table.		*Incubate for 24 h at 4° C on rocker table.
			\|
			Wash off 1° Antibody
	;*Wash off 1° Antibody		\|
	*Fill 3 containers with PBST		*Fill 3 containers with PBST
	*Put membrane in 1st PBST wash		*Put membrane in 1st PBST wash
Line 235:		Line 236:
	**5 min on rocker table		**5 min on rocker table
	*Rinse membrane in 3rd PBST wash		*Rinse membrane in 3rd PBST wash
			}}


	;*Prepare for 2° Antibody Incubation		{{2Box\|width1=300\|width2=300\|
			Prepare for 2° Antibody Incubation
			\|
	*Dilute 2° Antibody in PBSTb		*Dilute 2° Antibody in PBSTb
	**Ms 1:200		**Ms 1:200
Line 244:		Line 248:
	*Pour in 1° Antibody PBSTb mixture		*Pour in 1° Antibody PBSTb mixture
	*Incubate for 1 h at 4° C on rocker table.		*Incubate for 1 h at 4° C on rocker table.
			\|
			Wash off 2° Antibody
	;*Wash off 2° Antibody		\|
	*Fill 3 containers with PBST		*Fill 3 containers with PBST
	*Put membrane in 1st PBST wash		*Put membrane in 1st PBST wash
Line 258:		Line 262:
	**5 min on rocker table		**5 min on rocker table
	*Rinse membrane in diH2O		*Rinse membrane in diH2O
			}}

Bradley Monk: /* 5. MEMBRANE BLOCK AND ANTIBODY INCUBATION */

2013-05-19T00:56:54Z

5. MEMBRANE BLOCK AND ANTIBODY INCUBATION

@@ Line 187: / Line 187: @@
 *PBSTb = PBS + Tween + Milk Block
 *Use 5 g Carnation Powder Milk per 100 mL PBST
 {{Box|width=350px|PBST & PBSTb|
@@ Line 213: / Line 214: @@
 **Turn on rocker
 *Let incubate for 2 hours or overnight
 === ANTIBODY INCUBATION ===
-*Wash 4X/ 5min. with PBS-T.
+;*Prepare for 1° Antibody Incubation
-*Make 2° Antibody in PBS-T-Block.  (Ms 1:200)
+*Dilute 1° Antibody in PBSTb
-*Add membrane and incubate 1 hr.
+*Put membrane in plastic sealer bag
-*Wash 4X/5 min. with PBS-T.
+*Seal 3 sides of plastic
-*Wash 3X/ 5 min. with diH2O.
+*Pour in 1° Antibody PBSTb mixture
 [[File:SeeBlue Novex.png|350px]][[File:GluR234.jpg|350px]]

Bradley Monk: /* MEMBRANE BLOCK */

2013-05-19T00:45:59Z

MEMBRANE BLOCK

← Older revision		Revision as of 17:45, 18 May 2013
Line 212:		Line 212:
	*Put membrane in PBSTb container		*Put membrane in PBSTb container
	**Turn on rocker		**Turn on rocker
	*Let incubate for ~~1 hour~~		*Let incubate for 2 hours or overnight


	~~;Wash PVDF Membrane~~
	*Fill 2 ~~containers with PBST~~
	*Put membrane in 1st PBST wash
	**5 min on rocker table
	*Put membrane in 2nd PBST wash
	**5 min on rocker table
	*Rinse membrane with PBST



	=== ANTIBODY INCUBATION ===		=== ANTIBODY INCUBATION ===

Bradley Monk: /* 5. MEMBRANE BLOCK AND ANTIBODY INCUBATION */

2013-05-19T00:43:33Z

5. MEMBRANE BLOCK AND ANTIBODY INCUBATION

← Older revision		Revision as of 17:43, 18 May 2013
Line 183:		Line 183:
	===MEMBRANE BLOCK===		===MEMBRANE BLOCK===

	;Make ~~PBS-T~~ and Block Buffer (PBS-Tween & Milk Block)		;Make PBST and PBSTb Block Buffer
	*Use 5 g Carnation Powder Milk per 100 mL ~~PBS-T~~		*PBST = PBS + Tween
			*PBSTb = PBS + Tween + Milk Block
			*Use 5 g Carnation Powder Milk per 100 mL PBST

	{{Box\|width=350px\|~~PBS-T~~ & ~~Block~~\|		{{Box\|width=350px\|PBST & PBSTb\|
	;Step 1 - Make 1000 mL ~~PBS-T~~		;Step 1 - Make 1000 mL PBST
	*900 mL diH2O		*900 mL diH2O
	*100 mL PBS-10x		*100 mL PBS-10x
	*5 mL Tween-20		*1 mL Tween-20


	;Step 2 - Make 100 mL ~~PBS-T + Block~~		;Step 2 - Make 100 mL PBSTb
	*5 g Carnation		*5 g Carnation
	*100 mL ~~PBS-T~~		*100 mL PBST
	}}		}}
	{{Clear}}		{{Clear}}


	*~~Remove~~ membrane ~~from cassette~~		;Ready PVDF Membrane
	*~~Wash~~ membrane with ~~PBS-T 2X~~		*Dip membrane in MeOH
	*~~Place~~ membrane in ~~PBS-T-Block~~ for ~~1hr.~~		*Float membrane in PBST
	*~~Wash~~ membrane ~~2X/5min~~ with ~~PBS-T~~

			;Ready a Rocker Table for membrane blocking
			*Fill container with PBSTb
			*Put container on rocking table or shaker table
			*Put membrane in PBSTb container
			**Turn on rocker
			*Let incubate for 1 hour


			;Wash PVDF Membrane
			*Fill 2 containers with PBST
			*Put membrane in 1st PBST wash
			**5 min on rocker table
			*Put membrane in 2nd PBST wash
			**5 min on rocker table
			*Rinse membrane with PBST

Bradley Monk at 00:26, 19 May 2013

2013-05-19T00:26:19Z

← Older revision		Revision as of 17:26, 18 May 2013
Line 179:		Line 179:
	[[File:Blot Transfer.png]]		[[File:Blot Transfer.png]]

	==5. ANTIBODY ~~EXPOSURE~~==		==5. MEMBRANE BLOCK AND ANTIBODY INCUBATION==
	~~[[File:SeeBlue Novex.png\|350px]][[File:GluR234.jpg\|350px]]~~
	===~~ANTIBODY EXPOSURE STEP 1 -- MAKE~~ BLOCK ~~BUFFER~~===		===MEMBRANE BLOCK===

	;Make PBS-T Block Buffer (~~milk +~~ PBS + Tween)		;Make PBS-T and Block Buffer (PBS-Tween & Milk Block)
	*Use 5 g Carnation Powder Milk per 100 mL PBS-T		*Use 5 g Carnation Powder Milk per 100 mL PBS-T

	{{Box\|width=350px\|PBS-T-Block\|		{{Box\|width=350px\|PBS-T & Block\|
	;Step 1 - Make 1000 mL PBS-T		;Step 1 - Make 1000 mL PBS-T
	*900 mL diH2O		*900 mL diH2O
Line 193:		Line 193:


	;Step 2 - Make 100 mL PBS-T-Block		;Step 2 - Make 100 mL PBS-T + Block
	*5 g Carnation		*5 g Carnation
	*100 mL PBS-T		*100 mL PBS-T
Line 199:		Line 199:
	{{Clear}}		{{Clear}}

	~~===ANTIBODY EXPOSURE STEP 2 -- MEMBRANE BLOCK===~~

	*Remove membrane from cassette		*Remove membrane from cassette
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	*Wash membrane 2X/5min with PBS-T		*Wash membrane 2X/5min with PBS-T

	===~~ANTIBODY EXPOSURE STEP 3 --~~ ANTIBODY INCUBATION===

			=== ANTIBODY INCUBATION ===

	*Make 1° Antibody in PBS-T-Block.		*Make 1° Antibody in PBS-T-Block.
Line 220:		Line 221:
	Incubation overnight should be done in a cold room at 4o C		Incubation overnight should be done in a cold room at 4o C
	}}		}}

			[[File:SeeBlue Novex.png\|350px]][[File:GluR234.jpg\|350px]]

	{{Clear}}		{{Clear}}

Bradley Monk: /* BLOT TRANSFER STEP 3 -- RUN TRANSFER */

2013-05-19T00:20:45Z

BLOT TRANSFER STEP 3 -- RUN TRANSFER

← Older revision		Revision as of 17:20, 18 May 2013
Line 175:		Line 175:
	*lane 1 ladder is now on the right		*lane 1 ladder is now on the right
	*dip PVDF membrane in MeOH		*dip PVDF membrane in MeOH
	*dry on filter paper overnight		**(if stopping, dry membrane on filter paper overnight)

	[[File:Blot Transfer.png]]		[[File:Blot Transfer.png]]