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
messages.

Many thanks
                        Piero




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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