No replies to this topic

[craigGFFKR]

I am trying to identify novel protein-protein interactions with a transmembrane receptor in primary cells using immunoprecipitation and then SDS-PAGE with silver staining. The anti-receptor mAb I am using to pulldown the receptor has a lot of contaminants and leads to a high background, thus interfering with potential novel protein bands on the gel. I thought I could clean up the protein complex by using a tandem affinity approach in which I biotinylate cell surface proteins (including my receptor) before lysis, then do the immunoprecipitation as before with mAb and protein G resin, elute the complex from the resin, then pulldown with NeutrAvidin resin. The only issue with this is the elution from protein G resin. I usually use a low pH elution buffer to dissociate protein from IgG, but I believe the low pH elution would also dissociate the receptor-protein complex and I would lose the protein(s) of interest.

Is there an elution buffer I can use to preserve the protein-protein interactions but still release my protein complex from IgG?

Alternatively, is there a better way to go about all of this?

Thanks!