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Gal4 BD Antibody Problem

vuk savv at medizin.uni-leipzig.de
Wed May 16 11:05:31 EST 2001

Hi, Juan Zhou

I have a very similar problem, if not the same. I can not detect a Gal BD signal
with Clontech antibody. I have tried lots of different concentrations of primary
and secondary antibodies, Vectastain boosting, but no help - once I received
bands that could be at 22 kD, but when I tried to intensify with Vectastain, I
got all the non-specific bands. ETC.
Clontech people think my problem is the plasmid I am using - PGBT9. It has a
very low expression and is not detectable by Western blot according to Clontech
Have you checked your plasmid? I am switching to PGBKT7, with a stronger T7
promoter and higher expressivity, valued as +++ on Clontech scale (Yeast
Handling Catalogue, chapter Yeast Promoters). I will let you know if this helps.

Please if you have any new achievement on fighting this windmill of ours, let me
know, I could sure use it! Also, I would appreciate if you wrote about the
plasmid you are using, so I would know if other plasmids, with a higher
expressivity than PGBT9 , also create this kind of problems.
Vuk Savkovic

yuan zhou schrieb:

> After yeast two-hybrid, we identified several cDNAs that encode for
> proteins interacting with the bait.  Then we moved on to Western, hoping
> to see those proteins bind the bait in vitro.  First, we separated
> samples that contains AD-fused proteins, and then used the BD-bait as
> probe.  However, we could not detect any signal using BD mAb from
> Clontech.  The signal was missing even when the BD mAb was separated on
> gel, and then detected with  the anti-mouse IgG.  Has anyone encountered
> the same/similar problem?  Is the BD mAb not working?
> Best regards,
> Zhou

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