Immunoprecipitation of reduced proteins using Pierce kit - Immunoprecipitation using Pierce Kit (Nov/15/2006 )
Hi,
I am trying to immunoprecipitate a protein of my interest, and I'm using the Seize Primary IP kit from Pierce (Cat No.45335). The intention is to submit the immunoprecipitate to mass-spec protein sequencing and validate the specificity of the antibody. The antibody was self-developed, and the protein is a known protein (entire sequence available on NCBI)
The IP process using the Pierce kit involves this:
1. Couple the antibody to the gel - mix for 4hrs to overnight.
2. Add the protein sample to the antibody coupled gel - mix overnight.
3. Add the IP buffer, do a few washes (to remove the unbound proteins), and use the elution buffer to elute off the protein bound to your antibody in the antibody-coupled gel.
My problem is this:
I don't get any protein in the elute.
What I know about my antibody from western blots:
My antibody (self-developed) recognizes the protein only under denaturing & reducing conditions. It does not recognize the native protein, and does not recognize denatured but non-reduced (boiling and SDS, but no mercaptoethanol) protein. The protein sample I'm applying to my IP reactions is native protein - so it might as well be not bound and washed off during the wash steps. I'll do a western later to test if the washes/other filtrates contain my protein.
My problem is:
I can boil my protein sample and denature it, but not reduce it with mercaptoethanol, because I was told by Pierce tech support that if I did, when I apply such prepared sample to the antibody coupled gel, mercaptoethanol would mess up the antibody and render it functionless - so it might not bind to the protein anyways.
Can anybody suggest a way by which I can subject my protein sample to denaturing & reducing conditions and then immunoprecipitate it using my antibody? My intention is to submit such immunoprecipitated protein for mass-spec protein sequencing to confirm that my antibody is detecting its actual target protein, and not some non-specific product.
Any suggestions regarding techniques/kits would be greatly appreciated.
Thank you,
RR777
denature your protein (boil SDS mercapto), then get rid of mercapto by passing your protein on desalting column, and concentrate again on a centricon column. migrate in a native gel and try to detect your protein. if it works, you know you can IP it after getting rid of mercapto.
Hi Missele,
Thank you for your input. I will try what you've mentioned, to get rid of the mercaptoethanol and then concentrated the sample with centricon.
RR777
the problem is that I don't know how much the protein could "renature" in the absence of mercapto, that's why I suggest to check by blotting if the antibody still recognize.
This is a really naive advice.
This is a really naive advice.
My problem is this:
I don't get any protein in the elute.
This is what I said before - and that was based on Ponceau S staining on the membrane. I later did a western blot on all the four elutes I got after immunoprecipitation, and the filtrate (with the IP buffer) prior to the elution step, with a positive control (Protein samples I used for the IP assay). I got bands on all four elutes, with the band intensity being the highest in the first elute and the lowest in the last elute. The positive control worked and the filtrate also gave a strong band. I'm sure it's not just a washout of non-specific proteins in the elute, because the elute WB bands migrate at a lower MW than the positive control and the filtrate. So I think my IP worked, and now I have to figure out why I did not see the protein in my native gel in my previous WBs! I guess I'm gtting bands in the filtrate because maybe not all my protein is bound to the antibody-gel complex or I don't have sufficient antibody to capture all of my specific protein in the protein sample. I used 100ug of antibody for the assay.
Thank you for the inputs, though!!