Help w/ Ni column protein purification

Keith pitts at
Fri Jan 16 04:54:37 EST 1998

In our lab, we are using a hexa-his tagged protein overexpressed in a pET21b
vector (IPTG inducible).  I have tried to use the Novagen instructions for
binding the tagged protein to a Nickel column.  The protein doesn't seem to
stick well and comes out during elution with WASH buffer (60mM imidazole)
rather than the STRIP (100mM EDTA). Both are in 0.5M NaCl, 20mM Tris-HCl.

Has anyone run into problems w/ Ni columns.  Are there any suggestions I might
use for tweaking the protocol for greater protein adherence?

Thanks much,
(please cc to me if you reply via newsgroup posting)
Keith Pitts		_Lab Technician II_
pitts at

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