His-tagged protein won't stick to Ni column

Boris Steipe steipe at LMB.uni-muenchen.de
Wed Nov 20 04:09:42 EST 1996


> I've battled for well over a year with getting a His-tagged protein to bind
> to a Ni column.

This is most unusual. Assuming that you've tested every obvious
variation on the protocol, I would wonder whether the tag has not been
degraded off. You could attach a common peptide epitope like myc to the
tag (outside !) and check via Western blot whether the tag is still
there after expression (or buy the Dianova very expensive anti-His
monoclonal). Alternatively, there are a couple of very elegant
elternatives, S-tag, myc-tag itself, Flag-tag or the Strep-tag. See:

     Schmidt, T. G. M. & Skerra, A. (1994). One-step affinity
purification of bacterially
     produced proteins by means of the "Strep tag" and immobilized
recombinant core 
     streptavidin. J. Chromatography A 676, 337-345. 

Good luck,
Boris

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