I think one of the possible reasons why your protein loses activity is that
it is metal-dependent, and imidazole is a chelator. From this perspective
using another chelator (EDTA) would not help. I would consider recloning you
protein into another expression system, e.g., pMAL (NEB), where your protein
is fused to an affinity epitope (maltose-binding protein in case of pMAL)
that can be eventually removed by digestion with sequence-specific protease.
"soenke behrends" <behrends at plexus.uke.uni-hamburg.de> wrote in message
news:8tc5sp$9k0$1 at rzsun03.rrz.uni-hamburg.de...
> Dear netters,
>> 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