polyclonal antibody purification

ijiwaru at wheel.dcn.davis.ca.us ijiwaru at wheel.dcn.davis.ca.us
Tue Jan 28 04:22:14 EST 1997


In article <32eda9db.296595 at news.univie.ac.at>, a8803349 at unet.univie.ac.at
(Martin Offterdinger) wrote:

> On 26 Jan 1997 21:09:24 GMT, ageorge at casbah.acns.nwu.edu (ANNE GEORGE)
> wrote:
> 
> >I have been having problems trying to purify a polyclonal antibody raised in 
> >rabbits. The antigen was an e.coli expressed protein. There is a lot of
background.
> >does anyone out there know what is the best method.
> >Thanks
> >-- 
> >ANNE GEORGE
> >Northwestern University, Evanston, IL.   USA
> >ageorge at casbah.acns.nwu.edu
> >
> Dear Anne
> I would recommend affinity purification:
> Immobilise the purified protein on a column e.g.: HiTrap-NHS activated
> from Pharamacia, centrifuge the serum , dilute it 1:20 with PBS apply
> it onto the column and do an acidic elution with Glycine/HCl pH=2.7-
> afterwards the eluate has to be neutralised.
> Good luck Martin

Anne,

to add to the above, you might also consider changing the context of your
protein before coupling to insure that you get only those Abs specific for
your protein of interest.  You can also add a DEAE-Seph column step before
your antigen column to insure that your starting material is a bit
cleaner.

Lyle Najita
Plant Pathology
University of California - Davis



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