High MW IgG signals in IP/co-IP with protein G - (Feb/19/2014 )
I've been struggling with a co-IP for months and need some assistance. Namely, I'm detecting high molecular weight bands in my IgG IP control. I add 5ug of normal rabbit IgG (same as IP antibody), and perform the immunoprecipitation with protein G dynabeads. When I western blot, I get distinct bands at ~50, 75, and ~120 kDa with both light-chain specific HRP, and True-blot (a secondary reagent that only recognizes native IgG, supposedly). These bands make it very hard to tell if I get IP of target proteins ~60kDa and 140 in my actual sample lanes.
Standard SDS-PAGE and transfer to nitrocellulose. Nothing special.
Has anybody else had a tough time with normal rabbit IgG in regards to these high molecular weight species on western blot? A colleague in my lab also had the same trouble with this experiment.
I've detected similar bands (though not as robust) with BS3 cross-linking of the antibody or IgG to the dynabeads, so I'm wondering if this might be non-reduced/denatured or cross-linked protein-G/IgG complex eluting from the beads. Very annoying if so!
Any thoughts? I wold be happy to provide more info as needed.
Cheers and thanks!
Just in case anybody else runs into this problem - I figured it out. Embarrassingly simple problem, actually.
Turns out I was using insufficient reducing agent (an old/expired bottle). The high molecular weight signal was almost certainly nothing more than non-reduced IgG, as it cleared up with a new stock and higher concentration in my samples.