Immunoprecipitation problem - (Aug/27/2007 )
I'm having trouble with an immunoprecipitation experiment that is supposed to tie up my M.Sc., and I really want to get the problem nipped in the bud fast! I had so much trouble purifying my protein/antigen that I want to waste as little time on this new problem as possible.
My protein of interest (POI) was successfully induced and purified using NiNTA-6xHis tag technology under denaturing conditions (8M urea) and was dialyzed using a urea/pH gradient to 0M urea, pH 7.4. The protein sample has a lot of my POI in it based on SDS-PAGE/coomassie staining and it shows up right where it's supposed to on a western blot (~38 kDa). We did our assays for the activity that we were looking for and found that the activity was present, suggesting successful dialysis. Now, just as a tie-up to the experiments, we wanted to perform an immunoprecipitation to confirm that the activity seen was indeed due to our POI.
I'm using the Pierce Seize X Protein A Immunoprecipitation kit. I followed the directions exactly when generating the IP column, and used the DSS/DMSO to attempt to cross-link the primary antibody to the column (so that elution fractions should contain pure antigen rather than antigen/antibody complexes). I do up the IP experiment, and end up with a pre-load sample, a flowthrough, 3 wash samples, and 3 elutions.
Performing a western blot, I find that the IP was somewhat successful... I get bands for my POI in the pre-load (obviously) as well as the flow-through sample and the first elution sample.
Here's the problem: the band for my pre-load shows up as expected, ~38 kDa, whereas the bands in the FT and E1 samples are HUGE, like 250 kDa plus... running the samples on SDS-PAGE and Coomassie staining indicates that the proteins in the FT and E1 samples aren't even entering the resolving gel; in fact, the FT protein is still at the bottom of the well, and the E1 protein is stuck at the stacking/resolving gel interface.
So, what's up with this story? What could be causing this? I'm especially confused by the flow-through sample... I mean, after cross-linking the antibody with DSS/DMSO I made sure to follow all the wash steps stringently, such that there should have been no chemical residue left in the column, as well as a neutral (7.4) pH. So, just in the act of 'flowing-through' this IP column, my proteins are all aggregating/precipitating for some reason completely unknown to me.
P.S. Oh, and I should mention just for details' sake, that the antibody I'm trying to use for this is polyclonal. Shouldn't be a problem, should it?
P.P.S. At first I was thinking it might be antibody/antigen complexes coming out, due to unsuccesful cross-linking or something, but then i realized that this can't be the case... can it? I mean, aren't antibody/antigen complexes sensitive to SDS and would just be broken up by the SDS-PAGE?
No one has any ideas!?
What´s the composition of your binding buffer? Are you using reducing conditions in your SDS-PAGE sample buffer? Would it be possible to test whether the huge bands you see contain Igs? (for example, blotting only a secondary antibody specific for your IP antibody)