I'm trying to identify protein-protein interactions by doing an IP for my target protein and then identifiying interacting partners w/ MS. For optimization I've been doing WB to prove that I precipitate the target protein and silver stainings to see if additional proteins are pulled down.
For the IPs, I used Dynabeads Protein G (Invitrogen), but I get a huge signal from the antibody heavy and light chain in both the silver staining and WB. I'm guessing this would also be a problem w/ the MS so I would like to try coupling the antibody to the beads.
My question is whether I should try crosslinking the antibody to the protein G dynabeads, or would it be better to use NHS-acitvated magnetic beads that will directly covalently bind the antibody?
One of the concerns about the NHS-activated beads is that they would bind the antibody wherever there are primary amines, which could result in the lower affinity or avaliability of the Fab region for the antigen. Whereas with crosslinking to protein G, the antibody is already correctly positioned to interact w/ the antigen, which would result in a better precipitation (better meaning more protein)
Does anyone have any experience/thoughts/recommendations on the type of beads I should use?
Thanks a lot!