Thanks for all of the helpful suggestions. In reply to some of the
comments, I did purify by eluting with imidazole from a Ni-NTA column.
The imidazole I used is 99% pure, although I'm not sure whether it is
reagent or molecular biology grade. I also do use DTT in my lysis and
elution buffers, so these could contribute to the color.
After Ni-NTA, I dialyzed the protein into a buffer containing 10 mM Tris,
pH. 7.0, 10% Glycerol, as BME overnight, and then purified it on a
Q-column. I then did a final dialysis in buffer that contained 2 mM DTT
and 5 mM MgCl2 (although the color appears without the MgCl2 as well),
among other reagents. I would think that the two dialysis steps and the
Q-column would remove all of the imidazole. However, I did not run the
protein on a sizing column or use EDTA in any buffers. I could try using
a sizing column, and if that does not remove the color, I could use some
of the techniques suggested to determine if any metals are bound.
Matt
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