Help w/ Ni column protein purification

Vladimir Svetlov svetlov at
Fri Jan 16 10:27:05 EST 1998

In article <34BF2E5D.3ECB83A5 at>, Keith <pitts at>

> 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.

Never tried Novagen columns. Using Ni-NTA columns from Qiagen I noticed
that some proteins elute at lower Im concentrations than others, very
similar (like an internal deletions vs. full-length His-tagged
derivatives). Elution of a substantial amount of Ni-NTA-bound protein at 50
mM Im is not such a rare occurence. If this is a problem, I try to use 0
and 10 mM Im buffers for washing, and occasionally 1-2 M urea for general
clean-up if the purification is done at non-denaturing conditions (does not
unfold the proteins but disrupts many non-specific interactions). Elution
is done at 100-160 mM Im. 
And drop NaCl to 150 mM, too much salt is not good for you and for the
binding (although up to 2 M of NaCl can be used according to Qiagen, I
heard on number of occasions that high salt presented a problem during
isolation of one or another protein).

Vladimir Svetlov
McArdle Lab for Cancer Research
Dept. Oncology
1400 University Ave.
Madison, WI 53706

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