pull down troubles - (Jan/09/2008 )

Dear All
i am trying to confirm my Y2H screening by pull down
basically my prey is fused with GST(pET42a) and my bait has an His Tag (pET 29 vector)
i purified the bait on nickel column, and elute it.then i use the GST-prey stick to the beads ti pull it down....and detect the bait by specific antibody.
so fard i have signal everywhere, even with beads only.
I am wondering.
how much quantity of protein do you usually use for a pull down. i also read several papers with differents buffer...one with PBS, or Tris-EDTA or Hepes...i am using PBS (with mgcl2, DTT, bSA) so is there a better buffer to use for the binding reaction?
thanks for your answer
Camille

You may want to preclear the eluted fractions with glutathione bead before doing pull-down.
If your His-protein persists on binding to the glutathione bead, then change direction, using His-protein-beads to pull-down GST protein.

The binding buffer varies depend on the nature of your protein. So I can't say much about it. What about other people who work with your protein or similar previously? Which buffer did they use?