Unfortunately I don't know the answer to your question but I am
interested in the false positives you have picked up. Could you
tell me what kind of proteins this were? It could save me some time
Piero Morandini wrote:
> 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