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Questions about the 2-hybrid system

Celedonio Gonzalez cgonzale at magnus.acs.ohio-state.edu
Thu Jun 2 15:58:08 EST 1994


     Hi,
     I'm using the 2-hybrid system to study the dimerization of a protein, and
I'm having some results that I would like to comment:

     1.- Sometimes, after developing the plates with X-gal, I get a mixture of
white and blue colonies. All the colonies are supossed to be transformed with
the same two plasmids, so, how is it possible that same are producing
beta-galactosidase and some aren't?

     2.- In order to give numerical value to the strength of interaction
between the two fragments carried by the vectors, I've been assaying
beta-galactosidase for the transformants, but the results doesn't make much
sense. The activity can be very different even between two different
transformants from the same plate (like 5 time higher in one of them), being
both blue in the plate. Am I doing something wrong? Should I forget about
giving a numerical value to the interaction?

     3.- Some fragment A in one of the vectors gives blue colonies when
contransformed with the another vector containing the hole gen. But another
fragment B, which is bigger than (and contains) A, doesn't give blue colonies.
May be B doesn't fold in the right way, but then, can I conclude, in general,
that getting no blue colonies is evidence for the lack of interaction?

     Does any one out there have the same or similar problems? Sugestions are
needed!!.

        Celedonio



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