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purified two-hybrid proteins?

Piero Morandini pm1 at imiucca.csi.unimi.it
Tue Nov 4 11:41:31 EST 1997

Dear yeast netters,
after many false positives, I fished out of a two-hybrid library what seems
to be a true interactor of my pet protein.
Now it seems to me time to seek biochemical evidence for the interaction.
I suspect I will need for this decent amounts of the purified proteins.

Question is:  what is the feeling/experience of the people about this?
Should one first generate a clone for expression in coli or try to purify
it from yeast cells?
Is it better to keep the fusion protein as it is in the two-hybrid or
eliminate everything that is not coding for the interactor/bait protein?

I am using the pACT vector for the prey and I know that there is a
commercially available antibody against the GAL4 DBD.  Will this be enough
for the purification?

I had a look in the BioNet archives but it seems there isn't much on this
argument.  You can reply to me directly and I shall repost a digest of the

Many thanks

Piero Morandini
Biology Department "L. Gorini"
Section for Plant Physiology and Biochemistry
University of Milan
Via Celoria 26,  Torre 4C,
Milan 20133    Italy

tel:  +39-2-2660-4394                Email:  pm1 at imiucca.csi.unimi.it
fax:  +39-2-2660-4399

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