Smolke:Protocols/Western: Difference between revisions
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==Materials== | ==Materials== | ||
*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) | ||
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==Procedure== | ==Procedure== | ||
===Lysis=== | ===Lysis=== | ||
#Grow culture ( | #Grow culture (5mL works well) in appropriate (generally dropout) media | ||
#Pellet cells at 3000g, | #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 | ||
#Repellet | #Pellet cells at 3000g, 4 °C for 5 minutes | ||
# | #Reweigh tubes, resuspend at 100 uL/mg in 0.1 M NaOH | ||
# | #Incubate at RT for 5 min | ||
#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=== | ||
# | #Take 8uL prestained ladder (thaw and spin down - liquid often condenses on the lid) | ||
#Prep pre-cast 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 | ||
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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 | ||
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==References== | ==References== | ||
Kushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860. | |||
==Contact== | ==Contact== |
Revision as of 09:36, 28 June 2011
OverviewBlotting for large V5-tagged proteins in S. cerevisiae Materials
ProcedureLysis
SDS-PAGE
Semi-dry Transfer
Blotting
Notes
ReferencesKushnirov, V.V. Rapid and reliable protein extraction from yeast. 2000. Yeast 16: 857-860. Contact |