His-tag purification

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Thu Nov 7 20:56:20 EST 2002


> thank you very much,
You're most welcome.

> 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.

What exactly do you classify as 'failed' ? There are several  types of
problems with His-tagging, some of which hae specific solutions :) We have
not had too much luck with the strep-tag, but I know a few folks who like it
a lot. I hope it works for you.

> do you think an in vitro system is not suitable for purification with his
tag because of low amount of resulting tagged protein?

Depends what the purification is FOR. For crystallography or NMR - you need
ideally upwards of 50 mg of pure protein. This is still too expensive to do
in vitro, right now. Things may change. For smaller-scale applications in
vitro synthesis may be more applicable. I personally like to have TONS of
recombinant protein, to be able to do all sorts of studies - so it'd be a
while until I switch to in vitro for everything, however I am led to believe
that in vitro synthesis can help with difficult cases. Like any 'hot'
technology, it'd take a few years to truly emerge as something useful.

A.G.E.





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