immunoaffinity purification of a protein complex

redeamer alion85 at hotmail.com
Sat Jan 24 07:22:10 EST 2004


hbmao at u.washington.edu (Haibin Mao) wrote in message news:<63EC924E-457D-11D8-BD4A-000A95E527CA at u.washington.edu>...
> Hello,
> 
> We are trying to purify an endogenous protein complex from tissue by 
> immunoaffinity column. We want to elute the complex in its native form 
> as intact as possible in the absence of the antibody.  So far, we are 
> able to purify the full length protein of one subunit of this complex 
> in E. coli. We plan to generate polyclonal antibody against this 
> subunit and then make an affinity column. We plan to elute the complex 
> with the competition method using a lot of the E. coli purified 
> subunit. Since we have never done this kind of thing before, we are 
> wondering whether this approach sounds reasonable or not to the experts 
> out there.
> 
> Thanks.
> 
> Haibin Mao
> 
> ---

You will certainly need a conformational monoclonal antibody for that
purpose, if you want to have an effective purification of native
substance (complex)... i am running a similar project now...
monoclonal Abs will take some 2-3 months more than polyclonals, so it
is possible you have to wait quite  a bit..

As i understood you want the eukaryotic complex, so you ll have to
generate some eukaryotic protein for analysis of your monoclonals,
perhaps in Cos-7 cells (Cos is fine) or baculovirus system. Or quite
simpler but not so relevant: rabbit reticulocyte system (cell free)...

Good Luck, you have a lot of work to do :)


Drew



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