Smolke:Protocols/Western: Difference between revisions
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==Procedure== | ==Procedure== | ||
===Lysis=== | ===Lysis=== | ||
#Grow 25mL | #Grow culture (between 5 and 25mL works well) to saturation 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 supernatant, weigh, resuspend in water at 1 mg/uL | #Pour off supernatant, weigh, resuspend in water at 1 mg/uL | ||
#Transfer 100uL to a 1.5mL tube, repellet as before | #Transfer 100uL to a 1.5mL tube, repellet as before | ||
#Reweigh, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor) | #Reweigh, resuspend at 0.5 mg/uL in YPER (+EDTA-free protease inhibitor) |
Revision as of 15:23, 23 January 2009
Still a work in progress OverviewBlotting for V5-tagged proteins in S. cerevisiae Materials
ProcedureLysis
SDS-PAGE
Semi-dry Transfer
Blotting
NotesReferencesContact |