IP Problem

Arne Christensen via methods%40net.bio.net (by arnec from bio.umass.edu)
Thu Jul 10 15:02:35 EST 2008


Are there any antibodies raised against your protein in species other 
than rabbit (e.g. monoclonal, guinea pig)?  Using an antibody from a 
different species to detect would be the simplest solution.

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!
>
>
> Thanks,
>
> John
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>
>   


-- 

__

Arne K Christensen

Postdoctoral Research Associate
University of Massachusetts, Amherst

USGS Conte Anadromous Fish Research Center
One Migratory Way, PO Box 796
Turners Falls, MA  01376

Email: arnec from bio.umass.edu
Phone: (413) 863-3827
  Fax: (413) 863-9810
  URL: www.biobog.com



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