imidazol eluent alternative in his-tagged proteins

Nick Theodorakis nicholas_theodorakis at
Mon Nov 1 15:11:34 EST 1999

In article <381b0597.6810569 at>,
behrends at (Soenke Behrends) wrote:
> Dear netters,
> I am working on a hemoprotein where the heme is bound
> to the protein by a histidin (and specifically by the imidazol
> moiety). My impression and fear is, that after his-tag binding
> to a Ni column the elution with imidazol buffer not only
> destroys the protein column interaction but also frees
> my protein of heme. Does anyone know of an alternative
> way of elution (under native conditions) get the protein
> off the column.
> I have thought also to construct a  His-tag with TEV protease
> site and cleave it off the column, but I am forced to tag
> the C-terminus and I have not found any vector / technique
> to do that.
> Thanks a lot for any hint or comment
> Soenke

This isn't about alternative elution methods, but, since you have a
hemoprotein, it should be easy to detect if the heme becomes unbound
from the protein spectrophotometrically. Do an absorption scan of your
protein from about 400-500 nm in the absence or presence of various
concentrations of imidazole. If the heme group becomes unbound, the
absorbance peaks (esp. the Soret peak) should shift position
dramatically (at least, it does for hemoglobin and cytochrome c).


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