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Simple yeast lysis procedure

Leon Parker mrclean at U.WASHINGTON.EDU
Tue Aug 30 21:17:31 EST 1994

Can anyone suggest a quick and effective way for perparing whole cell
extracts of a plasmid-transformed S. cerevisiae strain (grown in CM media)
for Western blot analysis?  I've tried pelleting cells from 1 mL of log
phase culture, resuspending in 50 uL 2X SDS PAGE loading buffer, freezing
on dry ice (and I've tried liquid nitrogen), and then boiling for 5-10
minutes.  The yield of total protein using this method is very low
compared to the extracts I've prepared by smashing the cells with beads. 
Unfortunately, despite the addition of a plethora of protease inhibitors
and a considerable effort to keep things cold when I try glass bead
smashing method, my protein of interest always seems to be degraded when 
I analyze it by Western blotting.  I'm hoping a quicker, simpler method 
would eliminate this problem.  

Thanks in advance.  Please repond to me directly at: mrclean at u.washington.edu

--Leon Parker
Department of Microbiology
University of Washington

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