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Yeast Westerns

Nancy Lemieux nhl at binkley.cs.mcgill.ca
Sun Mar 13 14:52:52 EST 1994


HI

This is my first time reading this newsgroup.  I'm sure this is the right
place to ask.

I am trying to use the yeast 2-hybrid system to look for binding proteins. 
I am using the yeast strain Y190, and a plasmid with a GAL4 DNA binding
domain fused to my protein as a bait.  However, I am not able to detect the
expression of the protein.   I have tried all sorts of methods to detect by
Western (ECL with whole cell extracts, IP's.  Anti GAL4 antibodies,
followed by protein A crosslinked to  I 125, etc.)

Is it always hard to detect protein expression, or does the yeast strain
make a difference?

I know that the system works anyway because I used  E1A/Rb interactions as
a control, and could not detect their expression either, even though we
have excellent antibodies against these proteins.

Any suggestions? Please post here.


Nancy Lemieux
Dept. of Biochemistry
McGill University
Montreal, Canada
nhl at binkley.cs.mcgill.ca



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