histag elution alternative
krasel at wpxx02.toxi.uni-wuerzburg.de
Fri Oct 27 11:38:45 EST 2000
soenke behrends <behrends at plexus.uke.uni-hamburg.de> wrote:
> we purify a protein containing a HisTAG with
> a Ni-NTA matrix and elute the protein with
> imidazole (250mM). Unfortunately enzyme activity
> of the eluted protein is strongly inhibited by
> 250mM or 150mM imidazole, whereas 50mM imidazole
> lead to 50% of the activity without imidazole.
> We now want to turn to an alternative elution
> approach e.g. EDTA. Has someone made good ex-
> perience with this under native conditions and
> could provide us with a protocol?
Just putting 10 mM EDTA in your buffer of choice should be
sufficient. You will see the greenish Nickel colour go away
and the column turn white. Be aware that EDTA may change the
pH of your buffer.
You can afterwards re-charge your resin with NiCl2.
> I have also been thinking about whether one could
> use high concentrations of DTT.
DTT will turn your column into a dirty brownish colour.
Given that it is quite unclear what happens there (reduction,
but to what? I would guess that elementary Nickel is unlikely
to form), I would go with the EDTA elution.
A resin treated with DTT is almost impossible to recharge
(I know since I tried :-).
/* Cornelius Krasel, U Wuerzburg, Dept. of Pharmacology, Versbacher Str. 9 */
/* D-97078 Wuerzburg, Germany email: phak004 at rzbox.uni-wuerzburg.de SP4 */
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