Another IMAC question

Dr. Peter Gegenheimer PGegen at UKans.nolospamare.edu
Fri Aug 21 18:18:56 EST 1998


On Fri, 21 Aug 1998 13:51:50, James Fethiere <james at mpimf-heidelberg.mpg.de>
wrote:

> I am using a 1.6ml POROS Ni column to purify a protein eluted from a
> FLAG-M1 column with 2mM EDTA.  After a certain time I start to loose
> binding to the Ni column.
>
> I would like to know if it's possible that after approximately 1.5L of
> these 2mM EDTA preps, there is enough stripping of the Ni from the
> column to explain what I see.  I should mention that in the previous
> step of purification, EDTA is necessary to chelate Ca (3mM) that is
> required for binding of the protein to the FLAG M1 column.  Everything
> is done in 50mM Tris pH 8.

For the previous step, why not use another chelator? For example, EGTA binds
Ca(II) tightly, but Mg(II) about 1000X less well; I forget the affinity for
other metals. You should be able to identify a chelator which has binds Ca well
but has selectively reduced affinity for Ni or whatever metal is on your
column.

The reference for metal affinity constants is an article by Blanchard in Meth
Enzymol, vol 104.


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