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 _______________________________________________ Methods mailing list [email protected] http://www.bio.net/biomail/listinfo/methods
