Axin1 Co-IP - (Nov/26/2014 )

Hi,

I did co-ip on my protein lysate and pulled down with Axin1 antibody (Millipore, A5).  However, I can detect GSK3b binding to Axin1 (by western) it but I cannot detect axin1 both in the input and the immunoprecipate (IP) lysate. Has anyone experienced something similar?

The axin1 antibody is supposed to be very specific.  It was stored at the wrong temperature. Its supposed to be stored at 4deg but was accidentally stored at -20 for a few days.  Could this be the problem?

is the antibody rated for ip? it may be for denatured antigen (as in western blot), check on the data sheet that came with it.

did it work for the western after you froze it? if so then it probably wasn't significantly hurt by freezing.

If I understand your question correctly, you've performed an IP but do not see the target IP protein via western blot.  Yet you do see a "co-IP" interacting protein by western blot?  Is this correct?

1.  Are you using the same antibody for the IP and the western?  This can cause interference, but usually results in higher background and not a loss of signal.  It's possible that the antibody doesn't work for both processes, too.  I would generally recommend two antibodies for IP/coIP:  1 IP antibody and 1 western antibody for a given target antigen.

2.  What supports the axin1 antibody's specificity?  I would be leery of trusting some antibody suppliers' claims your own validation work if you run into problems.  It's possible for a dirty IP antibody to in fact pull down supposedly "co-IP'd" proteins (though I think GSK3 is known to interact with axin1, right?).  You need to know if your IP works first, in any case.