Smolke:Protocols/Western: Difference between revisions
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Still a work in progress | |||
==Overview== | ==Overview== | ||
Blotting for V5-tagged proteins in ''S. cerevisiae'' | |||
==Materials== | ==Materials== | ||
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==Procedure== | ==Procedure== | ||
===Lysis=== | |||
#Grow 25mL culture to saturation in appropriate (generally dropout) media. | |||
#Pellet cells at 3000g, 4°C for 5 minutes | |||
#Pour off supernatant, weigh, resuspend in water at 1 mg/mL. | |||
#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 | |||
#Pellet at 14000g, 4°C for 10 minutes | |||
===SDS-PAGE=== | |||
#Remove 40uL of lysate supernatant, mix with 10uL of 5x loading buffer | |||
#*Also take 5uL prestained ladder (thaw and vortex first) | |||
#Boil samples while prepping precast SDS-PAGE gel | |||
#Load 25uL of each sample | |||
#Run gel with MOPS running buffer, 150V, ~1hr | |||
===Transfer=== | |||
#Make 2x transfer buffer + 10% MeOH | |||
#Pre-equilibrate gel in 2x transfer buffer + 0.02% SDS, 10 minutes | |||
#Soak pags + membrane in 2x transfer buffer + 10% MeOH | |||
#Layer pad, membrane, gel, pad | |||
#Run 15V, 20 minutes | |||
===Blotting=== | |||
#Incubate membrane with 1x TBST + 5% milk, >1hr (rocking) | |||
#Wash twice with 1x TBST, 5 minutes (rocking) | |||
#Incubate membrane with 10mL 1x TBST + 5% milk + 2μL α-V5 antibody, >1hr (rocking) | |||
#Wash twice with 1x TBST, 5 minutes (rocking) | |||
#Mix 2.5mL each developing solution, add to membrane, incubate 5 minutes (rocking) | |||
#Image | |||
==Notes== | ==Notes== |
Revision as of 14:26, 23 January 2009
Still a work in progress OverviewBlotting for V5-tagged proteins in S. cerevisiae MaterialsProcedureLysis
SDS-PAGE
Transfer
Blotting
NotesReferencesContact |