Immunoprecipitation and immunohistochemitry help required

Ian A. York iayork at panix.com
Mon Mar 3 11:02:23 EST 1997


In article <Pine.HPP.3.95q.970303151045.12253B-100000 at ssa.bris.ac.uk>,
S.D. Wainwright <ogsdw at ssa.bris.ac.uk> wrote:
>
>I have a monoclonal antibody (IgG2b) raised against a peptide
>and it immunoblots very well. However, it does not immunopreciptiate
>from TX100 lysates and I have had very weak immunoprecipitation results
>from 2% SDS cell lysates diluted to 0.1% SDS.  The antibody does not 

An antibody which blots but doesn't IP, and which IP's weakly with a more
strongly denaturing detergent, suggests that it may be seeing a
non-donformational epitope which is hidden in the conformed protein.  You
may be able to use this in IPs by fully denaturing your protein.  The 2%
SDS approach was worth a try; since that didn't work, you might try
something like heating your sample before the IP (try a couple of
conditions; heating to 75oC for 30 minutes has worked for me).  Of course,
this may cause your protein to precipitate or something awful like that,
and you may just have to settle for an antibody which doesn't IP.

Good luck.

Ian
-- 
      Ian York   (iayork at panix.com)  <http://www.panix.com/~iayork/>
      "-but as he was a York, I am rather inclined to suppose him a
       very respectable Man." -Jane Austen, The History of England



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