R. Woodward wrote:
>> Dear All
> We are having a few problems with the yeast two hybrid system
> (Clontech Matchmaker 2). The problem is that we cannot appear
> to get any positive interactions even with proteins and protein
> fragments that I know interact e.g. G-protein subunits although the
> control provided with the kit works o.k. I am using growth on
> dropout -His and beta galactosidase colony lift assays in yeast strain
> Y190. are there any proctical problems that I should take into account?
> Has anyone any experiences with this system (good or bad).
>> Thanks very much
>> Email rw200 at cus.cam.ac.uk>> --
I would absolutely agree with your experience that positive controls
sometimes do not give positive results if you had used HF7c or derived
strains. But in Y190 I never experienced loss of positivity. Are you
sure your inserts are in frame and do not contain stop codons? (Did you
completely sequence your inserts?) Are you sure your inserts are
expressed? (Did you check this with antibodies?). If both is true maybe
your fusion protein does not fold correctly. (Did you clone various
regions of your protein?).
Molecular Human Genetics