His-tag purification

Artem Evdokimov AEVDOKIMOZ at cinci.rr.com
Thu Oct 31 22:14:07 EST 2002


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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