(by jleonard.iii from gmail.com)
Thu Jul 10 12:43:03 EST 2008
I'm looking to perform an IP Western of a protein that runs at around
50-55kD on an SDS-PAGE gel - approximately the same as my antibody's heavy
chain. As such, it could be very difficult to detect presence of my protein
by IP Western, since the antibody is generally eluted along with the
antigen. I have read protocols which suggest to cross-link your antibody to
the protein A/G beads prior to IP, however I'm using rabbit antiserum (not
immunoaffinity purified), so I don't know whether this will be effective.
A co-worker of mine suggested running a non-denaturing gel instead to
observe either a) separation of the protein of interest and the antibody by
nature of different charge/size ratios; or b) differential shifting of
the antibody band (when compared with a negative/positive controls) to
indicate interaction with my protein of interest. I don't know how
effective this would be, but in that case I would need a good NON-DENATURING
elution buffer to detach my antigen from the bead complex.
The whole purpose of this experiment is to determine whether my antiserum
will be sufficient for IPs, ChIP, etc or whether I should immunoaffinity
purify it. (I'll be pre-clearing with normal rabbit serum to reduce
If anyone has comments/suggestions about this, or knows of A GOOD
NON-DENATURING ELUTION BUFFER, I'd love to hear it!
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