Problems with IPs - (May/23/2012 )
I have been trying to co-IP two transcription factors from nuclear fractions. Up until today, I have had varying success with the 'trap' protein, but almost NEVER see my original "bait" protein that I IP'd for. I can clearly see it in the same samples that have not been IP'd, and since I can see heavy chain and some interacting partners, it appears to have IP'd, but I can't get it to show up on an immunoblot. Problem number 2 is that now my trap protein appears to me migrating 20 kDA higher in the IP samples than in the untreated lysates. Any ideas on either front?? Thanks!
Verify that the 20 kDa higher protein is actually the one you are looking for - use a peptide competition assay.
Run a sample of the pre-clearing beads on a gel - it may be that your protein is sticking to the beads not the Ab.