Immunoprecipitate of big proteins

P.C. PC at no.email.sorry
Wed Dec 3 22:19:14 EST 2003


looks like your protein is asking for a tag...
you might try FLAG- or HA- tag. If your protein is cytoplasmic, then 
anti-HA would normally give cleaner IPs.

Ian A. York wrote:
> 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 




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