purified two-hybrid proteins?

[Jan Carette]

Dear Pierro,

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
in screening.

Thank You,

Jan Carette

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
> 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