I have been working with the yeast two-hybrid system for a year now trying
to determine specific subunit interactions within an oligomeric protein.
As an internal control for my protein I have made 4 constructs of two
separate subunits that we know interact in the holoprotein but I have been
unable to pick up any interaction in the two-hybrid. I am using the
Clonetech pGBT9 and pGAD424 vectors. Initially in trying to quantitate the
beta-gal activity I thought perhaps simply the problem was substrate
sensitivity, as I can get colonies to grow on triple drop out plates. But
I can not get any signal (beta-gal activity) even using the most sensitive
luminescent substrate (AMPGD). My first question is that perhaps b/c the
ADH promoter is truncated there is not enough expression to measure
beta-gal activity?..although I was assured that w/ AMPGD as the substrate
this would not be the case. Secondly, should I just resolve myself to the
fact that perhaps some interactions just cannot be identified in the
two-hybrid and move on?
If anyone has any suggestion or experinces they would like to share it
would be appreciated. In addition, I am tempted to clone my constructs
into high expressing vectors although my boss is not keen on investing in
the Clonetech pACT2/pAS2 vectors when I am unable to get the first set to
work. Would anyone out there happen to have available and ofcourse be so
kind to share any gal based two-hybrid vectors that are not copyrighted
and are high expressing?
Thanks so much.
nayers at utmem1.utmem.edu