Ab purification problem

Dr Engelbert Buxbaum engelbert_buxbaum at web.de
Fri Oct 18 03:32:01 EST 2002


"D.K." wrote:

> - Purify IgG on Protein A, elute with 100 mM Glycin, pH 2.5,
> immediately neutralize to pH 8.0 with 2 M Tris, pH 9.0,
> add NaCl to 0.35 M final.
> - Run over an antigen column, load 2 times to ensure saturation
> of binding sites, with 3.6 M MgCl2, pH 6.5. Dialyse extensively,
> make sure to leave room for water in dialysis bag. MgCl2
> elution is a lot milder on most native proteins than standard
> acid elution.

Elution with caotropes from an affinity column selects for mediocre
antibodies, as the realy high affrinity ones stick or elute at
concentrations of denaturants, which are destructive.

Electroelution might be the better alternative.



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