(by allison from nospam.com)
Thu Jul 10 15:33:39 EST 2008
John Leonard wrote:
> Hello all,
> 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
> non-specific binding).
> If anyone has comments/suggestions about this, or knows of A GOOD
> NON-DENATURING ELUTION BUFFER, I'd love to hear it!
If you run a regular SDS gel without reducing agent in the sample buffer
(ie, no beta-mercaptoethanol) then the antibodies will run at 150K. If
your protein is a single unit then it will still run at about 50-55K.
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