Smolke:Protocols/Western - Revision history

Josh K. Michener at 16:36, 28 June 2011

Josh K. Michener — Tue, 28 Jun 2011 16:36:48 GMT

←Older revision		Revision as of 16:36, 28 June 2011
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	==Materials==		==Materials==
-	*Y-PER ([http://www.piercenet.com/products/browse.cfm?fldID=06010430 Pierce])
-	*Halt EDTA-free Protease Inhibitor ([http://www.piercenet.com/Products/Browse.cfm?fldID=02040806 Pierce])
	*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX)		*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX)
	*Prestained protein ladder (NEB P7711S)		*Prestained protein ladder (NEB P7711S)
Line 22:		Line 20:
	===Lysis===		===Lysis===
	#Grow culture (5mL works well) in appropriate (generally dropout) media		#Grow culture (5mL works well) in appropriate (generally dropout) media
-	~~#Pellet cells at 3000g, 4°C for 5 minutes~~
	#Pre-weigh an appropriate number of eppendorf tubes		#Pre-weigh an appropriate number of eppendorf tubes
	#*The variation in weight of the eppendorf tubes is significantly larger than the weight of the pellet		#*The variation in weight of the eppendorf tubes is significantly larger than the weight of the pellet
-	#~~Pour off the supernatant~~, resuspend in ~~1mL water~~. ~~Transfer to a pre-weighed~~ 1~~.5mL tube~~	+	#Pellet cells at 3000g, 4 °C for 5 minutes
-	#Repellet ~~as before~~, ~~reweigh, and~~ resuspend at ~~0.5 mg/~~uL in ~~YPER (+EDTA-free protease inhibitor)~~	+	#Reweigh tubes, resuspend at 100 uL/mg in 0.1 M NaOH
-	#~~Agitate~~ at RT for ~~20min~~	+	#Incubate at RT for 5 min
-	#~~Pellet~~ at ~~14000g for 10 minutes~~	+	#Repellet cells, resuspend at 25 uL/mg in 1x Laemmli buffer and transfer 50uL to a PCR tube
		+	#Heat at 95 °C for 3 min
		+	#Repellet, max speed, RT, 5 min
		+	#Remove supernatant
		+	#*Samples can now be stored at RT overnight

	===SDS-PAGE===		===SDS-PAGE===
-	#~~Remove 20uL of lysate supernatant, mix with 5uL of 5x loading buffer~~	+	#Take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid)
-	~~#*Also take~~ 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid)	+	#Prep pre-cast SDS-PAGE gel
-	#~~Boil samples while prepping precast~~ SDS-PAGE gel	+
	#Load 20uL of each sample		#Load 20uL of each sample
	#Run gel with MOPS running buffer, 150V, ~1hr		#Run gel with MOPS running buffer, 150V, ~1hr
Line 72:		Line 72:

	==References==		==References==
		+	Kushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860.

	==Contact==		==Contact==

Josh K. Michener at 19:47, 23 July 2010

Josh K. Michener — Fri, 23 Jul 2010 19:47:36 GMT

←Older revision		Revision as of 19:47, 23 July 2010
Line 21:		Line 21:
	==Procedure==		==Procedure==
	===Lysis===		===Lysis===
-	#Grow culture (~~between~~ 5mL works well) in appropriate (generally dropout) media	+	#Grow culture (5mL works well) in appropriate (generally dropout) media
	#Pellet cells at 3000g, 4°C for 5 minutes		#Pellet cells at 3000g, 4°C for 5 minutes
-	#Pour off the supernatant, resuspend in ~~0.5mL~~ water. Transfer to a pre-weighed 1.5mL tube	+	#Pre-weigh an appropriate number of eppendorf tubes
		+	#*The variation in weight of the eppendorf tubes is significantly larger than the weight of the pellet
		+	#Pour off the supernatant, resuspend in 1mL water. Transfer to a pre-weighed 1.5mL tube
	#Repellet as before, reweigh, and resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)		#Repellet as before, reweigh, and resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)
	#Agitate at RT for 20min		#Agitate at RT for 20min
-	#Pellet at 14000g~~, 4°C~~ for 10 minutes	+	#Pellet at 14000g for 10 minutes

	===SDS-PAGE===		===SDS-PAGE===
	#Remove 20uL of lysate supernatant, mix with 5uL of 5x loading buffer		#Remove 20uL of lysate supernatant, mix with 5uL of 5x loading buffer
-	#*Also take ~~12uL~~ prestained ladder (thaw and ~~vortex first~~)	+	#*Also take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid)
	#Boil samples while prepping precast SDS-PAGE gel		#Boil samples while prepping precast SDS-PAGE gel
	#Load 20uL of each sample		#Load 20uL of each sample
Line 64:		Line 66:
	==Notes==		==Notes==
	*SDS-Page: At this step I usually take 40uL of supernatant (and mix it with 10uL 5x loading buffer), then remove the rest and resuspend the pellet in 200uL 60% glycerol. Then I take 40uL of the resuspended pellet and mix it with 10uL 5x loading buffer and then continue following the protocol. This pellet resuspention isn't optimized; the pellet fraction usually ends up looking like a dot/blob when the western is imaged - but you can at least get an idea of where your protein is in the cell (supernatant = soluble, pellet = probably membrane bound). -Isis		*SDS-Page: At this step I usually take 40uL of supernatant (and mix it with 10uL 5x loading buffer), then remove the rest and resuspend the pellet in 200uL 60% glycerol. Then I take 40uL of the resuspended pellet and mix it with 10uL 5x loading buffer and then continue following the protocol. This pellet resuspention isn't optimized; the pellet fraction usually ends up looking like a dot/blob when the western is imaged - but you can at least get an idea of where your protein is in the cell (supernatant = soluble, pellet = probably membrane bound). -Isis
		+	**For membrane-associated proteins, I sometimes resuspend the pellet in 2% SDS, boil it, spin it down again, and load the supernatant. That can pull out weakly associated proteins.

	*Semi-Dry Transfer: I just use 1x transfer buffer (with no methanol) and don't pre-equilibrate the gel. I think I can get away with this because my proteins are relatively small (at least compared to Josh's). Also, for some reason the voltage doesn't always stay at 15V after I set it, so I always go back and double check and adjust after a few minutes of running. -Isis		*Semi-Dry Transfer: I just use 1x transfer buffer (with no methanol) and don't pre-equilibrate the gel. I think I can get away with this because my proteins are relatively small (at least compared to Josh's). Also, for some reason the voltage doesn't always stay at 15V after I set it, so I always go back and double check and adjust after a few minutes of running. -Isis

Josh K. Michener at 19:07, 26 March 2010

Josh K. Michener — Fri, 26 Mar 2010 19:07:11 GMT

←Older revision		Revision as of 19:07, 26 March 2010
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	==Procedure==		==Procedure==
	===Lysis===		===Lysis===
-	#Grow culture (between ~~5 and 25mL~~ works well) in appropriate (generally dropout) media	+	#Grow culture (between 5mL works well) in appropriate (generally dropout) media
	#Pellet cells at 3000g, 4°C for 5 minutes		#Pellet cells at 3000g, 4°C for 5 minutes
-	~~#*The idea is to lyse equal masses of cells in each tube. You need a large initial culture volume in order to measure the initial pellet mass.~~	+	#Pour off the supernatant, resuspend in 0.5mL water. Transfer to a pre-weighed 1.5mL tube
-	#Pour off supernatant~~, weigh~~, resuspend in water ~~at 1 mg/uL~~	+	#Repellet as before, reweigh, and resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)
-	#Transfer ~~100uL~~ to a 1.5mL tube~~, repellet~~ as before	+
-	~~#Reweigh~~, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)	+
	#Agitate at RT for 20min		#Agitate at RT for 20min
	#Pellet at 14000g, 4°C for 10 minutes		#Pellet at 14000g, 4°C for 10 minutes

	===SDS-PAGE===		===SDS-PAGE===
-	#Remove ~~40uL~~ of lysate supernatant, mix with ~~10uL~~ of 5x loading buffer	+	#Remove 20uL of lysate supernatant, mix with 5uL of 5x loading buffer
-	#*Also take ~~5uL~~ prestained ladder (thaw and vortex first)	+	#*Also take 12uL prestained ladder (thaw and vortex first)
	#Boil samples while prepping precast SDS-PAGE gel		#Boil samples while prepping precast SDS-PAGE gel
-	#Load ~~25uL~~ of each sample	+	#Load 20uL of each sample
	#Run gel with MOPS running buffer, 150V, ~1hr		#Run gel with MOPS running buffer, 150V, ~1hr
	#*Run until dye front is near bottom of gel		#*Run until dye front is near bottom of gel
Line 65:		Line 63:

	==Notes==		==Notes==
-	SDS-Page: At this step I usually take 40uL of supernatant (and mix it with 10uL 5x loading buffer), then remove the rest and resuspend the pellet in 200uL 60% glycerol. Then I take 40uL of the resuspended pellet and mix it with 10uL 5x loading buffer and then continue following the protocol. This pellet resuspention isn't optimized; the pellet fraction usually ends up looking like a dot/blob when the western is imaged - but you can at least get an idea of where your protein is in the cell (supernatant = soluble, pellet = probably membrane bound).	+	*SDS-Page: At this step I usually take 40uL of supernatant (and mix it with 10uL 5x loading buffer), then remove the rest and resuspend the pellet in 200uL 60% glycerol. Then I take 40uL of the resuspended pellet and mix it with 10uL 5x loading buffer and then continue following the protocol. This pellet resuspention isn't optimized; the pellet fraction usually ends up looking like a dot/blob when the western is imaged - but you can at least get an idea of where your protein is in the cell (supernatant = soluble, pellet = probably membrane bound). -Isis

-	Semi-Dry Transfer: I just use 1x transfer buffer (with no methanol) and don't pre-equilibrate the gel. I think I can get away with this because my proteins are relatively small (at least compared to Josh's). Also, for some reason the voltage doesn't always stay at 15V after I set it, so I always go back and double check and adjust after a few minutes of running. -Isis	+	*Semi-Dry Transfer: I just use 1x transfer buffer (with no methanol) and don't pre-equilibrate the gel. I think I can get away with this because my proteins are relatively small (at least compared to Josh's). Also, for some reason the voltage doesn't always stay at 15V after I set it, so I always go back and double check and adjust after a few minutes of running. -Isis
		+	**On the high voltage source, you set upper limits on both the current and voltage. For the semidry transfer you have (initially) a very low voltage and very high current. Make sure the current limit is wide open, and the voltage limit is what's keeping the voltage at 15V. If the current limit is initially responsible for keeping the system at 15V then the voltage will drift up over time. -Josh

	==References==		==References==

Josh K. Michener at 19:02, 26 March 2010

Josh K. Michener — Fri, 26 Mar 2010 19:02:24 GMT

←Older revision		Revision as of 19:02, 26 March 2010
Line 9:		Line 9:
	*Halt EDTA-free Protease Inhibitor ([http://www.piercenet.com/Products/Browse.cfm?fldID=02040806 Pierce])		*Halt EDTA-free Protease Inhibitor ([http://www.piercenet.com/Products/Browse.cfm?fldID=02040806 Pierce])
	*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX)		*NuPAGE Novex Bis-Tris 4-12% mini gels (Invitrogen NP0321BOX)
-	*Prestained protein ladder (~~Invitrogen LC5800~~)	+	*Prestained protein ladder (NEB P7711S)
	*Protein loading buffer		*Protein loading buffer
	*MOPS buffer (Invitrogen NP0001)		*MOPS buffer (Invitrogen NP0001)

Isis Trenchard: /* Notes */

Isis Trenchard — Tue, 20 Oct 2009 22:53:46 GMT

Notes

←Older revision		Revision as of 22:53, 20 October 2009
Line 65:		Line 65:

	==Notes==		==Notes==
		+	SDS-Page: At this step I usually take 40uL of supernatant (and mix it with 10uL 5x loading buffer), then remove the rest and resuspend the pellet in 200uL 60% glycerol. Then I take 40uL of the resuspended pellet and mix it with 10uL 5x loading buffer and then continue following the protocol. This pellet resuspention isn't optimized; the pellet fraction usually ends up looking like a dot/blob when the western is imaged - but you can at least get an idea of where your protein is in the cell (supernatant = soluble, pellet = probably membrane bound).

		+	Semi-Dry Transfer: I just use 1x transfer buffer (with no methanol) and don't pre-equilibrate the gel. I think I can get away with this because my proteins are relatively small (at least compared to Josh's). Also, for some reason the voltage doesn't always stay at 15V after I set it, so I always go back and double check and adjust after a few minutes of running. -Isis

	==References==		==References==

Josh K. Michener: Smolke:Western moved to Smolke:Protocols/Western

Josh K. Michener — Tue, 20 Oct 2009 21:05:24 GMT

Smolke:Western moved to Smolke:Protocols/Western

←Older revision

Revision as of 21:05, 20 October 2009

Josh K. Michener at 21:32, 21 July 2009

Josh K. Michener — Tue, 21 Jul 2009 21:32:44 GMT

←Older revision		Revision as of 21:32, 21 July 2009
Line 23:		Line 23:
	#Grow culture (between 5 and 25mL works well) in appropriate (generally dropout) media		#Grow culture (between 5 and 25mL works well) in appropriate (generally dropout) media
	#Pellet cells at 3000g, 4°C for 5 minutes		#Pellet cells at 3000g, 4°C for 5 minutes
		+	#*The idea is to lyse equal masses of cells in each tube. You need a large initial culture volume in order to measure the initial pellet mass.
	#Pour off supernatant, weigh, resuspend in water at 1 mg/uL		#Pour off supernatant, weigh, resuspend in water at 1 mg/uL
	#Transfer 100uL to a 1.5mL tube, repellet as before		#Transfer 100uL to a 1.5mL tube, repellet as before

Josh K. Michener at 23:30, 19 February 2009

Josh K. Michener — Thu, 19 Feb 2009 23:30:24 GMT

←Older revision		Revision as of 23:30, 19 February 2009
Line 1:		Line 1:
	{{Smolke_Top}}		{{Smolke_Top}}
-		+	{{TOCright}}
-	~~Still a work in progress~~	+

	==Overview==		==Overview==

Josh K. Michener at 21:44, 25 January 2009

Josh K. Michener — Sun, 25 Jan 2009 21:44:39 GMT

←Older revision		Revision as of 21:44, 25 January 2009
Line 4:		Line 4:

	==Overview==		==Overview==
-	Blotting for V5-tagged proteins in ''S. cerevisiae''	+	Blotting for large V5-tagged proteins in ''S. cerevisiae''

	==Materials==		==Materials==
Line 22:		Line 22:
	==Procedure==		==Procedure==
	===Lysis===		===Lysis===
-	#Grow culture (between 5 and 25mL works well) ~~to saturation~~ in appropriate (generally dropout) media	+	#Grow culture (between 5 and 25mL works well) in appropriate (generally dropout) media
	#Pellet cells at 3000g, 4°C for 5 minutes		#Pellet cells at 3000g, 4°C for 5 minutes
	#Pour off supernatant, weigh, resuspend in water at 1 mg/uL		#Pour off supernatant, weigh, resuspend in water at 1 mg/uL
Line 36:		Line 36:
	#Load 25uL of each sample		#Load 25uL of each sample
	#Run gel with MOPS running buffer, 150V, ~1hr		#Run gel with MOPS running buffer, 150V, ~1hr
		+	#*Run until dye front is near bottom of gel
		+	#Crack open gel, trim off top (wells) and bottom of gel with razor
		+	#*Can trim gel down further if all lanes weren't used

	===Semi-dry Transfer===		===Semi-dry Transfer===
		+	#Cut out membrane slightly larger than gel
		+	#*Be careful not to touch membrane - use forceps.
	#Make 2x transfer buffer + 10% MeOH		#Make 2x transfer buffer + 10% MeOH
	#Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes		#Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes
	#Soak pads + membrane in 2x transfer buffer + 10% MeOH		#Soak pads + membrane in 2x transfer buffer + 10% MeOH
	#Layer pad, membrane, gel, pad		#Layer pad, membrane, gel, pad
		+	#Roll a pipet over stack to press out bubbles
	#Run 15V, 20 minutes		#Run 15V, 20 minutes
		+	#*Check for transfer - did the (prestained) ladder transfer over?

	===Blotting===		===Blotting===
	#Incubate membrane with 1x TBST + 5% milk, >1hr (rocking)		#Incubate membrane with 1x TBST + 5% milk, >1hr (rocking)
		+	#*Make sure there are no clumps in the milk (particularly a problem if you store the TBST+milk)
	#Wash twice with 1x TBST, 5 minutes (rocking)		#Wash twice with 1x TBST, 5 minutes (rocking)
	#Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking)		#Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking)
		+	#*Can go overnight at 4°C for higher sensitivity
	#Wash twice with 1x TBST, 5 minutes (rocking)		#Wash twice with 1x TBST, 5 minutes (rocking)
	#Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)		#Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking)
-	#Image	+	#Take membrane out, place on plastic wrap, blot lightly with Kimwipe. Then fold over plastic wrap to cover (squeeze out bubbles)
		+	#Image membrane
		+	#*Use gel imager on chemiluminescence setting. Set for 5-100 s, in 5s increments
		+	#*It's also useful to take another picture in white light (without moving membrane) to image the ladder.

	==Notes==		==Notes==

Josh K. Michener at 22:23, 23 January 2009

Josh K. Michener — Fri, 23 Jan 2009 22:23:45 GMT

←Older revision		Revision as of 22:23, 23 January 2009
Line 22:		Line 22:
	==Procedure==		==Procedure==
	===Lysis===		===Lysis===
-	#Grow ~~25mL~~ culture to saturation in appropriate (generally dropout) media.	+	#Grow culture (between 5 and 25mL works well) to saturation in appropriate (generally dropout) media
	#Pellet cells at 3000g, 4°C for 5 minutes		#Pellet cells at 3000g, 4°C for 5 minutes
-	#Pour off supernatant, weigh, resuspend in water at 1 mg/uL.	+	#Pour off supernatant, weigh, resuspend in water at 1 mg/uL
	#Transfer 100uL to a 1.5mL tube, repellet as before		#Transfer 100uL to a 1.5mL tube, repellet as before
	#Reweigh, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)		#Reweigh, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor)