If there is a polyclonal Ab for your target, I'd suggest IP-ing with that instead since they recognize multiple epitopes and increase the chance you'll pull the target. Also check what antigen your mAb was raised against to make sure it isn't only recognizing an internal sequence of the polypeptide that is only exposed under denaturing/reducing conditions. You also might want to add 0.1% Triton and/or SDS to your sample and let it incubate 20m-1hr before IPing as that will help break up protein complexes and might give your mAb more access to the target epitope.

Are you doing a direct IP (incubate Ab with beads, then bead-Ab with sample) or an indirect IP (incubate Ab with sample, then sample-Ab with beads)?  I've generally had better luck with the indirect method, but it all depends on your target protein and the Ab you're using.  Abcam had a pretty good webinar on the topic a couple years ago, I've attached the slides from that for your reference

Hello Toni, 

Thank you so much for the response. 

We may be able to obtain the polyclonal Ab for the Synthase; however, because our goal is to see what Ags this particular MAb binds in the stool. Though, having the polyclonal Ab as control would be nice. Do you think that if the Poly does pull down but the MAb does not, then it is not the IP method fails, but perhaps the binding of MAb is not strong enough in IP? 

This MAb is specific for virus envelope. I am just testing whether it may cross-react with bacterial antigen from stool. That is a good point. We have the sequence of the bound protein obtained from mass spec of the Coomassie gel band earlier, so I will do a sequence analysis.

From earlier denaturing Western, we know that the MAb bound only in a reduced stool condition because positive band was seen in Reduced and not Non-reduced. That was why in one of the experiments, I reduced the stool before incubation with the coupled beads using 1X Reducing Reagent (used in regular Western blot), then did several buffer exchange to rid the RA from MAb, and also nothing was positive. 

How do we know if the MAb's epitope is exposed only after some conformational changes due to some thing else bind to it, especially when we are not even sure if the MAb actually binds to Synthase? In other words, do you think that (1) running the lysate on membrane, (2) blot Mab in Western to obtain positive band, and (3) mass spec the gel band could prove that indeed the MAb bind to that Ag? I guess, I can also try co-IP (well, if I can get the IP to work first)? Is there any way besides crystallography? I am going to try native gel next week. 

I am currently using the Pierce Crosslink Magnetic IP/Co-IP Kit (https://www.lifetechnologies.com/order/catalog/product/88805). So, the cell lysate had been diluted in their IP Lysis buffer which does contain 1% NP40 (and 25 mM Tris, 150 mM NaCl, 1 mM EDTA, , and 5% glycerol). I will try the 0.1% Triton and SDS in addition later when I repeat the IP again soon. Do you think we can ever over-denature the proteins by adding multiple detergents? Just so I know, detergents like SDS, NP40 or Triton-X100 denature (eliminate the 3D structure of proteins, and thus help to generate their monomeric versions), but do not reduce the proteins, right?

I have so far tried direct IP. That is a good suggestion; I will try that later. But, even though the bead is supposed to bind to Fc of Ab, do you think lysate (binds to Fab) may still interfere with the bead coupling and therefore the efficiency of IP pull-down?

Thank you so much for your thoughtful response. Hope to get back to you soon with more updates. Have a good night.