His-tag purification

c9520071 at hotmail.com c9520071 at hotmail.com
Wed Nov 6 08:46:09 EST 2002

thank you very much,

i tried optimizing salt- and Imidazol concentration. everything failed. now i´ll attach an streptag II to my proteins and hope to get better results.
do you think an in vitro system is not suitable for purification with his tag because of low amount of resulting tagged protein?

i hope i´ll get better results next time...

cheerz, christoph

Artem Evdokimov wrote:

> Ni-NTA purification will inevitably give you some background. In order to
> *minimize* background (note the emphasis on minimize) you can optimize your
> pH and salt concentration (experimentally, of course) - the result would be
> individual for a specific protein. You also should try binding the protein
> to the beads in ~20 mM imidazole - this often reduces the background
> dramatically. Also, you should try to maximize the protein/resin ratio - it
> will help compete out junk.

> In the end, if everything else fails you can follow your first step of
> purification with some sort of secondary step.

> A.G.E.


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