Smolke:Protocols/Western: Difference between revisions
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==Procedure== | ==Procedure== | ||
===Lysis=== | ===Lysis=== | ||
#Grow culture (between | #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 | ||
#Pour off the supernatant, resuspend in 0.5mL water. Transfer to a pre-weighed 1.5mL tube | |||
#Pour off supernatant | #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, 4°C for 10 minutes | ||
===SDS-PAGE=== | ===SDS-PAGE=== | ||
#Remove | #Remove 20uL of lysate supernatant, mix with 5uL of 5x loading buffer | ||
#*Also take | #*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 | #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 | ||
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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). | *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== |
Revision as of 12:07, 26 March 2010
OverviewBlotting for large V5-tagged proteins in S. cerevisiae Materials
ProcedureLysis
SDS-PAGE
Semi-dry Transfer
Blotting
Notes
ReferencesContact |