Smolke:Protocols/Western: Difference between revisions
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==Procedure== | ==Procedure== | ||
===Lysis=== | ===Lysis=== | ||
#Grow culture ( | #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 | #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 | #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 | #*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 | ||
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==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 |
Revision as of 12:47, 23 July 2010
OverviewBlotting for large V5-tagged proteins in S. cerevisiae Materials
ProcedureLysis
SDS-PAGE
Semi-dry Transfer
Blotting
Notes
ReferencesContact |