immunoaffinity purification of a protein complex
Walter Schick - BioSepCo
walterschick at astound.net
Sat Jan 24 09:46:57 EST 2004
Try www.genwaybio.com, and ask them their technical opinion. Their IgY
immunoaffinity columns have different properties from IgG, and may work
better for your complex.
----- Original Message -----
From: "redeamer" <alion85 at hotmail.com>
To: <methods at hgmp.mrc.ac.uk>
Sent: Saturday, January 24, 2004 4:22 AM
Subject: Re: immunoaffinity purification of a protein complex
> 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 :)
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