native HisTag elution alternative
behrends at plexus.uke.uni-hamburg.de
Fri Oct 27 05:53:15 EST 2000
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?
I have also been thinking about whether one could
use high concentrations of DTT. In the manual
(Quiaexpressionist) they write that DTT can reduce
Nickel thereby preventing nickel from binding His-tagged
proteins. High concentrations of a reducing agents
would be good for the protein anyway, but it is not
mentioned as an elution strategy.
I am not so keen on low pH for elution, since I fear
that it will not be any good for the protein. But I
have so far no evidence on that.
We would really appreciate any comment or tip
Thanks for your time and help
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