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
Department of Microbiology
University of Washington