IgG heavy chain from IP - (May/05/2013 )
Hi, I have done a few immunoblots and i'm trying to understand why I'm getting several bands: I have done an IP of 1 specific protein, then I run this sample on a gel and transfer. Then i incubate with primary antibody, which is the same antibody as I did the IP with (this is a control lane), as I'm blotting against the same protein as the IP'd protein. it is a rabbit monoclonal antibody and i then incubate with secondary anti-rabbit IgG. I have spoken to a girl in the lab and she says it is the heavy (and a faint light) chain of the IgG. I was wondering if anyone could shed any light on why this would show up on the blot/if there is any literature on this.
So - a normal western detection works like this with regards to the antibodies: protein>primary antibody>secondary antibody OK?
So when you do an IP you are pulling down with an antibody and removing the protein and bound antibody from the beads - this is then denatured and run on a gel - so the antibody is still there in the form of heavy and light chains, now the secondary antibody you use during the detection can bind to those (the primary shouldn't).... If you want to get around this you need to use a primary from a different species to the one you did your IP with. You can also try using protein G-HRP conjugates in the place of a secondary, as these should bind well to the primary antibody.