pAS2 expression and HA antibodies

Steve Elledge selledge at bcm.tmc.edu
Sun Oct 29 12:32:26 EST 1995


Dear 2 hybers,

	The ability to see a protein expressed from pAS2 with HA antibodies has no 
correlation with success in 2 hybrid screening.  Only about 5 or 10% of our 
fusions can be detected by HA western, although they can usually be 
detected by polyclonal sera if you happen to have it for the protein of 
interest.  The abundance of the fusion protein can vary for a number of 
reasons including stability of RNA, protein, or translatability of the 
protein.  The important issue is that you have enough protein to fill up 
all of the GAL4 binding sites in the genome, which requires very few 
molecules, probably in the hundreds or less.   I believe they estimate that 
there are  approximately 10 molecules of GAL4 in a yeast cell under 
represed conditions, and more in the presence of galactose, but the 
important point is that it is a low number.  Too much GAL4-DNA binding 
domain fusion protein might even be deleterious theorectically because 
protein not bound to DNA competes for activation domain fusion protein with 
the protein bound in front of the reporter genes.  Soooooo, don't worry too 
much about not seeing your protein by western.  If the fusion junction is 
correct by sequence, go for it and good luck.

Steve Elledge





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