My antibody does not work for IP, but works well fpr western? - antibofy not working for IP (Dec/06/2007 )
After tried 5 antibodies, I finally find a rabbit Poly that works very well for western blotting, but my ultimate goal is IP.
It DID NOT work for IP? is there a way to improve the performance of the AB on IP? It is quite difficult to find another Ab.
I used protein G agarose beads. the protocol and reagent worked in the past with several differnt antibodies, including rabbit polyclonal Abs. I run a western after the IP, there are some heavy chain visible, but much weaker compare to the IgG control... I wondered why the antibody isn't working? I can try protein A agarose beads as well if the Ab has low affinity with the protein G beads. 
strange.
polyclonal rabbit IgG should be recognized by protein G (as much as by protein A)
if your problem is that protein G is not recognizing your antibody, try CnBr activated sepharose (you crosslink the antibody to the sepharose)
the problem may be that the polyclonal antibody may be against an epitope that is not exposed in the native protein. have you tried an elisa against the native protein? you may need a different antibody.
"the problem may be that the polyclonal antibody may be against an epitope that is not exposed in the native protein"
I saw you posted many useful replys on this forum! Respect! I like your logo too "I work for what I paid for..."
Will you please explain more that why an Ab works well for western but not for IP? It gives a signle, cleam, and and wide band with only 20 ug total proteins on western.
Thanks a lot,
Sparrow
I am using a salmon sperm DNA Protein G agarose beads for ChIP. I don't know if the bead you mentioned will work for me. I think it should...
chip, chip, chip, chipping sparrow
hi,
could be the antibody recognizes a linear peptide, but in ur native protein form only part of the peptide or even less than that is available for ur antibody which cud explain the week signal!!!
I saw you posted many useful replys on this forum! Respect! I like your logo too "I work for what I paid for..."
Will you please explain more that why an Ab works well for western but not for IP? It gives a signle, cleam, and and wide band with only 20 ug total proteins on western.
Thanks a lot,
Sparrow
I assume for western you are first running SDS-PAGE, that will denature your protein and break its 3D structure. This expose epitopes that are otherwise protected (i.e. non-accessible to antibody because of protein conformation). For IP your protein is in native state (non-denature) so these epitopes will not be accessible for the antibody.
as mdfenko suggested you can easily find this out by ELISA. you can actually compare native protein to denatured one this way by boiling some of your protein before coating the plate. If your antibody does not recognize native protein you'll have to try find a different one. If your antibody recognizes both native and denature, your problem is in the IP conditions.
hope this helps.
in addition to what others have written...
many times antibodies are made against fragments of a protein rather than the whole. the fragment that is used to produce the antibody may be from a portion of the protein that is folded inside (hence, not exposed). so even though the antibody is polyclonal, the epitope (fragment) that it was made against may not be exposed in the native configuration.
I wonder if for co-ip, it shouldn't be monoclonal? It certainly wouldn't give unspecific background. With polyclonal, you can't be so sure if you co-ip the right thing, if the later step is spectrometry.
Yeah, It's for IP though, besides, unfortunately, the protein I am working with is not well studied, as a result, only polyclonals are commercially available.
It will not worth the trouble to develop our own antibody either