Two-Hybrid Problem

PAULD at CLONTECH.COM PAULD at CLONTECH.COM
Tue May 2 21:13:57 EST 1995


DEZWAAN_T at a1.mscf.upenn.edu (Todd DeZwaan) wrote:
>In article <hindges-280495162309 at 130.60.120.11>,
hindges at vetbio.unizh.ch
>> I am using pGBT9 for producing my protein as a fusion with the GAL4
>> BD. But I can't detect any fusionprotein in westerns loading about 
>> 40ug of protein extract on the gel. I sequenced of course the 
>> construct to proove in frame cloning. Any suggestions of more 
>> experienced user of this system would be appreciated.

> Well, to give a shorter answer, I do use the pGBT9 plasmid for my >
fusion and we couldn't detect anything either.  We sequenced the >
plasmid and everything was fine.  We have gone ahead and used it for >
two-hybrid selections and have pulled things out of library screens that >
would be expected to interact. A colleague of mine has said that the >
pGBT9 expresses at lower levels than the original Fields vetors so this >
might explain a lack of signal in a Western. Bottom line is it is working.

>Colin

We have confirmed that pGBT9 has a level of protein expression much
lower than pAS2.  This seems to be due to the version of the ADH1
promoter used in its construction.  The pAS2 plasmid has ca. 600 bp
more of the 5'-end of the ADH1 promoter.  Antibodies which easily detect
the GAL4-BD fusions expressed from the pAS2 do not always detect
pGBT9 or its derivatives, despite the fact that the same pGBT9 fusion
has been shown to interact with activation domain fusions in the
Two-Hybrid assay.   We are currently looking at methods to enrich the
yeast lysates for the presence of the GBT9 derived GAL4-BD fusion
proteins.

Paul Diehl, Ph.D.
CLONTECH Technical Service
800-662-2566 x 141
pauld at clontech.com




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