imidazole and degradation

Cornelius Krasel krasel at wpxx02.toxi.uni-wuerzburg.de
Wed Jun 23 03:29:32 EST 1999


Eva Horak <EM_Horak at fccc.edu> wrote:
> We were wondering if anyone out there has experienced degradation of
> their his-tagged protein by high concentrations of imidazole (250 mM for
> example) when eluting from a nickel column?  Is there better way to
> elute proteins from these columns to avoid degradation?

It is very unlikely (if not impossible :-) that the imidazole causes
the degradation of your protein. You can try to elute with 10 mM EDTA
instead, but you have to recharge your column with NiCl2 afterwards
because the EDTA works by complexing the Ni ions.

However, the best strategy for avoiding protein degradation is:

1) work in the coldroom
2) work fast
3) use protease-depleted bacterial strains (e.g. BL21 or lon- strains)
4) use protease inhibitors

(in range of decreasing importance, IMO).

--Cornelius.

-- 
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany   email: phak004 at rzbox.uni-wuerzburg.de  SP4 */
/* "Science is the game we play with God to find out what His rules are."  */



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