remove antibody from eluate in co-immunoprecipitation - (Dec/14/2011 )
I'm running a co-immunoprecipitation experiment in which I used quite a lot of protein extract and Flag-agarose beads. I eluted with the flag peptide but even with this I know I have quite a lot of antibody eluted and present in my sample. Before loading the SDS-PAGE for cutting bands for MS analysis I would like to remove the antibody. Anybody know a good way to do this? I'm affraid the IgG will be very intense in my gel after staining and will hide other proteins. However, I don't want to manipulate my sample a lot to avoid protein losses. Any piece of advice is welcome!!
I think GE Healthcare has a kit for IgG removal (prior to 2D PAGE). Otherwise you could use Prot A or G. Depends of course on the animal source of your Igs.
Are you using preconjugated flag-agarose beads or are you conjugating them yourself? Have you actually checked the amount of antibody in your eluate? I might expect the antibody elution may not be so much if you are using the preconjugated beads since they do a cross-linking that is more stable than the general antibody-binding strength.