Antibody sticks to antigen affinity column

WS via methods%40net.bio.net (by novalidaddress from nurfuerspam.de)
Sat Mar 6 08:15:39 EST 2010


Dear experts,

I try to purify a chicken IgY by affinity chromatography. The hapten
(a small "chemical" molecule, not a protein or peptide) is covalently
coupled to sepharose.

In order to find appropriate conditions, I applied 1mg of IgY (diluted
in 100mM tris, pH 7.5) to the column and almost no protein ran
through. I tried elution with 2M MgCl2, 5M urea and 100mM gylcine, pH
2.5 successively, washing with 100mM Tris between the steps. I got
some small peaks by monitoring the eluate for UV absorption, but the
fractions do not contain much protein, so I conclude that the majority
of the desired antibody still sticks to the column. Maybe it's of
importance that the column has been designed for bulk purification,
the capacity is expected to be about 100mg in respect to IgY, so the
load for my tests is very low.

Any ideas what else I could try? The eluted IgY should be functional,
of course.

Many thanks for your help!

Wo


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