Smolke:Protocols/Western: Difference between revisions
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==Overview== | ==Overview== | ||
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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 ( | *Prestained protein ladder (NEB P7711S) | ||
*Protein loading buffer | *Protein loading buffer | ||
*MOPS buffer (Invitrogen NP0001) | *MOPS buffer (Invitrogen NP0001) | ||
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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 | ||
# | #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 | |||
#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). -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 | |||
**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== | ||
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 |