Two Hybrid Problem

Colin Rasmussen crasmussen at anat.med.ualberta.ca
Sun Apr 30 21:10:46 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. Thank you.

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





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