Polyclonal Ab versus Monoclonal Ab for immunoaffinity chromatography - (Jul/25/2006 )
Ok, here goes....
So, I've been trying to purify a protein for the past few months with little success using His-tag/Ni-NTA methods. I get my protein, but unfortunately it's just not clean enough for my purposes, as quite a few background proteins seem to bind to the Ni-NTA resion and come out as well (using an E. coli expression system). Was going to use gel filtration to try to separate my protein out from the rest, until the unfortunate discovery that one of the impurities coming out with my protein is pretty much the exact same size as my protein of interest.
At this point, I have speculated using immunoaffinity chromatography to try to separate my protein from the rest in the POST-His purifications (so most bacterial background proteins have already been removed). My supervising professor seems to be quite keen on trying the immunoaffinity chromatography using cyanogen bromide-activated Sepharose 4B to immobilize our antibody for purifying the protein.
My conundrum is this: the antibody we have is polyclonal, derived from a short peptide (not sure how short, but I think in the range of 10-15 amino acid residues) from the N-terminal region of the protein I'm trying to purify. Most immunoaffinity chromatography experiments I have seen always use monoclonal Ab, and while my supervising prof wants to give this a try, another prof on my supervisory committee (who is more of a protein expert than my supe) seems to think this would be a waste of time and money since its polyclonal and not monoclonal. I did not have time to get this person to clarify the reasoning behind this for me before they left on holidays, etc., so I thought I'd come on here and see what kind of insight I could could into this situation.
What concerns are at play here that my supervisory-committee prof was alluding to, that would make this a waste of time and/or money? Can this type of immunoaffinity purification of a protein be done using polyclonal antobody? If so, what sort of things should I pay attention to and be concerned about using a polyclonal antibody as opposed to a monoclonal antibody?
Any help anyone could give me regarding this would be MUCH appreciated! Thanks in advance.
pretty long story ahh!!!!!
come to the point,
advantages i cud see
1,using immuno affinity chromatography to purify ur protein of interest after niNTA purification.
2,u raised Ab against a PEPTIDE (hopefully which will not allow low affinity antibodies)
questions i want to ask
1, how immunogenic is ur peptide, is it quite good one (if s u can expect specific antibodies)?
2,did u purify ur pAb with affinity purification (if s then u can go for next step, if not u shud check)
1, if u purified ur pAb with peptide affinity column, then check the reactivity of pAb towards native state and denatured stated via western blotting.
2, if u see good reactivity in denatured state n not in native state, i wud not suggest u to go for next step.
3, if it reacts with both structures of ur protein, then i m pretty sure that u can have success
questions for my curiosity
1, wat reason wud u give for co purification of nonspecific protein, is it because the non specific protein interacts with ur protein of interest or binding directly to niNTA column?
if the second case is true then increase the immidizole concentration in washing steps n give a try!
i hope u got every thing clear
if not let me know