Expression library screening with fusion proteins
nantel at biotech.lan.nrc.ca
Tue Aug 15 13:54:11 EST 1995
<Pine.ULT.3.91.950814150135.6767C-100000-100000-100000-100000 at zombie.oul
"Petri T. Kursula" <pkursula at zombie.oulu.fi> writes:
> I am starting to look for proteins that bind a specific protein domain
> that we are producing as a GST fusion protein. I wonder if anyone has any
> experience in screening an expression library with such a fusion protein,
> using anti-GST anibody and a secondary antibody coupled with alkaline
> phosphatase to visualize the positive clones, i.e. the ones that bind the
> fusion protein.
> What kind of conditions should one use to make the fusion
> protein bind the ones on the filter made from the expression library
> plates? Also, how easy would you think it would be to totally wash such a
> filter so you could use a different fusion protein to screen the same
> filter? What kind of conditions should you absolutely NOT use? Any help
> will certainly be appreciated!
A lot of this depend of the type of protein that you plan to use as a
probe. Anmyway, I'd be surprised to see where you hope to find (and pay
for) enough anti-GST antibody for such a screen. I would rather suggest
using the pGEX-2tk vector which contains a phosphorylation site for the
commercially available cAMP-dependent kinase from heart muscle. The
exact labeling and screening conditions are in a 1993? Biotechniques
paper by Ron & Haberer.
Personnaly, I've successfully used biotinylated MBP-fusion proteins to
isolate plant genes encoding novel bZIP factors. The exact protocol
should be out by now in Plant Molecular Biology Reporter.
Biotech Research Institute
National Research Council Canada
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