Co-IP Befuddlement

[Terror Tabby]

I’ve been struggling to get this co-IP to work for months now. Let me start off by saying that the interaction between these two proteins has been demonstrated before. Another lab where I work did some of these co-IPs and it has worked for them an in the same cell type I am using. I used the exact same lysis buffers and IP procedure as they recommended but nothing has worked for me so far. I told this other lab that the co-IPs were not working but they didn’t have any other ideas to try that I have not tried already. My PI will not let my give them my samples to try even thought they offered.

Only one thing works for me, I can IP for protein A and then probe for protein A on a Western. At least I know that my antibody is binding to the beads. This IP was very clean too, the only other band I saw was for the light chain.

I am using protein G Dynabeads from Invitrogen. I have tried 1ug antibody/50ug of sample protein to 4ug antibody/50ug of sample protein. When I incubate my lysate with the beads bound to the antibody, I take 50ug of the lysate and dilute it in 200ul of IP lysis buffer. Invitrogen recommends that I incubate the beads with my sample at RT for 20min, which I have done and I have incubated as long as overnight at 4C. I my samples in 20ul and I load all 20ul in one well. I have tried IPing for protein A and probing with B and vice versa. There is another protein C that also interacts with A. I have tried that too. Proteins A, B and C are abundant in my cells. The antibodies I have chosen are widely used and are rated for Westerns and IPs.

IP Lysis Buffer Contains
50mM Hepes [pH 7.4], 100mM NaCl, 2 mM EDTA, 0.5% NP-40, 10% Glycerol, 0.1% Tween 20, 100uM DTT, and protease inhibitors.

Washing Buffer
1xPBS and 0.02% Tween

Elution Buffer
2X sample prep buffer or 50mM Glycine + 2x sample prep buffer