Immunoprecipitate of big proteins

D.K. dk at no.email.thankstospam.net
Wed Dec 3 18:16:20 EST 2003


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
>>
>> proteins, it seems not efficiently and also it's not good for
>> histostaining. Can someone give me
>>
>> suggestings to efficiently pull dowm complexes with different lysis
>> buffer. Thanks!
>>
>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.

I agree. 

This is routibe for monoclonals and apparently also happens even 
with polyclonals. We had a case where, for the same 145K protein, 
polyclonals derived from inclusion bodies immunization were just 
about useless in IPs while immunization with baculovirally produced 
protein gave several great batches of serum. (Proteolytic fragments 
very different for denatured vs native for some proteins)? 

DK




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