IP Problem

AK via methods%40net.bio.net (by akhan357 from sbcglobal.net)
Thu Jul 10 17:42:06 EST 2008


I had same problem once added with my co-IP protein died upon heating the 
sample. 6M urea in sample buffer did the trick without heating which sent 
the IgG bands close to 100kDa. if I remember correctly I also added 10mM DTT 
that apparently did not effect shift of IgG to higher mol wt signal. good 
luck.
AK
"John Leonard" <jleonard.iii from gmail.com> wrote in message 
news:mailman.701.1215716798.3533.methods from net.bio.net...
> 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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