<a href="http://www.bio.net/hypermail/sponsor.html"><img src="http://www.bio.net/hypermail/sponsor.gif"></a><br>bionet.molbio.methds-reagnts archives: HELP!---Immunoaffinity column doesn't work

Nikolai Chitaev nikolaic at VISAR.WUSTL.EDU
Fri Mar 8 15:11:48 EST 1996

Tanja wrote:
> To: methods at net.bio.net
From: Jan Schroeder <jschroed at vzdmza.zdv.uni-mainz.de>
Subject: HELP!---Immunoaffinity column doesn't work
> Date: 6 Mar 1996 11:20:54 GMT
> Hi there,
> I am quite new to handle with Immunoaffinity Chromatography and I
> want to purify a protein from Hordeum vulgare with this subject.
> Before I start to purify the protein. I have to clean the polyclonal
> mouse IgG with a protein A column (BioRad, Affi-Gel Protein A G=
> el).

Why do you want to purify your protein on IgG column?
What happends if your polyclonals mainly IgM or IgA which are
specific to your protein of interest?
Solutions: Use coupling reagents which will attach all classes of Igs 
or repeate immunization untill you get a secondary responce.

> The antibody did not bound to the protein A and I found the
> complete IgG in the first wash step. Then I tried a different way:
> I tried to couple the IgG via caboanhydrate moities to hydrazide
> activated agarose beats (BioRad, Affi-Gel Hz Immunoaffinity Column)=
> In the first step I detected again the complete IgG so it did not
> bind to the gel.
> Maybe there are eome hints behind the product informations?
> Please contact Jan Schroeder<jschroed at vzdmza.zdv.uni-mainz.de>
> Thanks, Tanja

Look also in Pierce catalogue, quite informative.
Good luck


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