westerns from yeast

Archa Fox a.fox at biochem.usyd.edu.au
Sun Dec 14 23:02:38 EST 1997


Hi everyone,

I am using yeast for one hybrid and two hybrid screens.  I am having
troubles getting specific bands for the Gal4 DNA binding Domain in a
western.  I've tried two different antibodies, one monoclonal and one
polyclonal and both give lots of background bands, exactly the same
pattern as in my negative control - for all my samples.  I am pretty
sure that my yeast protein extraction method is good enough, I get a
nice range of bands on a gel and also, it is good enough to see single,
specific bands with my Gal4 activation domain antibody and the
corresponding plasmid (pGAD10).  (I have tried three different
extraction methods though including TCA and Ureas/SDS)

I've also tried three different expression vector systems, thinking that
the plasmid wasn't expressing the protein at high enough levels to be
detectable - pGBT9, pYX232 and pAS2-1.  These don't seem to make a
difference.

Does anyone have any ideas? 
Thanks in advance,
Archa Fox




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