<Pine.3.89.9408301930.A27546-0100000 at carson.u.washington.edu>
mrclean at U.WASHINGTON.EDU (Leon Parker) writes:
> 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.
Greetings from Berkeley!! The lysis procedure you may want to have a
look at is that of alkaline lysis. The reference is
Lyons S; Nelson N.
An immunological method for detecting gene expression in yeast
Proceedings of the National Academy of Sciences of the United States
America, 1984 Dec, 81(23):7426-30.
And the method described therein can also be applied to making extracts
from cells in liquid culture. Together with a potent protease inhibitor
such as described in Aris and Blobel (Methods in Enzymology 194) this
should eliminate proteolysis much better than the standard
SDS/glass-beads based methods. Drawback is, it is slower, especially if
you include a TCA precipitation step. Advantage is, it is quantitative
and you do not have to account for loss.