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.
Dept. of Biochemistry
nhl at binkley.cs.mcgill.ca