Immunoprecipitate of big proteins

Ian A. York iayork at panix.com
Wed Dec 3 19:30:44 EST 2003


In article <ibrssvkkgodqr8vqb8gfkv34nef4kbiv94 at 4ax.com>,
D.K.  <dk at no.email.thankstospam.net> wrote:
>On Wed, 03 Dec 2003 21:31:40 GMT, "Kyle Legate" <legatek at hotmail.com> wrote:
>>zhang184 at umn.edu wrote:
>>> My antibodies work well in western blot to detect the 120kD proteins.
>>> But when I IP and blot the
>>>
>>It sounds like you need a different antibody. Antibodies that work on
>>Western but not IP or histological work generally recognize an epitope in
>>the denatured state, but not in the native state.
>
>This is routibe for monoclonals and apparently also happens even 
>with polyclonals. We had a case where, for the same 145K protein, 

If you're desperate, and don't have an antibody that recognizes native 
protein, you might be able to get something by denaturing, then IPing.  
I've done this by heating the sample to 70 oC before the IP.  This is far 
from generally satisfactory, of course, for several reasons.  If you're 
looking for complexes (co-precipitations) naturally they'll fall apart as 
you denature.  Many proteins will precipitate as they heat up; even if 
yours doesn't, be sure to clarify the sample after heating.  But as I say, 
if you're desperate, it might be a workaround.

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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