Yeast two-hybrid vectors

Rohini R Deshpande deshpand at UMICH.EDU
Wed Jan 22 09:46:02 EST 1997


I have the pACT2 and pAS2-1 vectors that I don't mind sharing with you. I
had very good interaction and high beta-gal activity using the Y190 yeast
strain.
-Rohini
Univ of Michigan, Ann Arbor.

On Tue, 21 Jan 1997, nancy ayers wrote:

> Hi,
> 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.
> Nancy
> nayers at utmem1.utmem.edu
> 
> 




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