Q: stabilizing enzyme during storage

Philipp Wechner philipp.wechner at uibk.ac.at
Mon Jul 1 02:37:49 EST 2002


I would rather put the EDTA to the sample after elution. EDTA strips the
nickel from the column - you then have all the ni in your sample (there is
enough of it in the sample even if you elute without EDTA). I had problems in
the past with such ni-contaminated samples.

and another point: you elute with 20 mM imidazole? I normally do the washing
with 20 mM and then go to 250 mM imidazole for elution. I think that you have
very "unclean" protein in your sample if you elute with this low
concentration - is there any HIS-taged protein in your "elution" at all? in
my opinion it is still on the column after treatment with 20 mM imidazole....



Mauricio Alberto Realpe-Quintero schrieb:

> I have heard of increasing stability of 6XHis purified proteins through
> the addition of EDTA to the final elution buffer, however this should deal
> with the protein activity and correct folding in a case-based
> matter.  Perhaps you can try.  Have you ?.
>
> Luck,
>
> Mauro
>
> Mauricio Realpe. M.Sc.
> Ph.D. student.  Institute of Biotechnology/IBT (www.ibt.unam.mx)
> Universidad Nacional Autónoma de México/UNAM, Cuernavaca. Mexico.
> Tel. 52 555 622 7612.   Fax 52 777 317 2388
>
>         ¨The only way to succeed in anything is to give EVERYTHING¨
>
> On Sun, 30 Jun 2002, Pedro Rocha wrote:
>
> > Hello,
> >
> > I would appreciate any input from protein experts on how to stabilize a
> > purified
> > protein (obtained by overexpression in E.coli).
> >
> > This particular enzyme is purified using Ni-NTA resin, being eluted with
> > 20 mM
> > imidazole, 300 mM NaCl and 50 mM NaH2PO4. The enzyme is fully functional
> >
> > immediately after purification. However, it is very unstable. Storing,
> > even for a
> > few hours at 0-10 C  (ice or fridges) knocks-out most of the activity.
> > Same for
> > -20C. Addition of various %s of glycerol did not help at all. I would
> > greatly
> > appreciate any suggestions of additives that could be tried to stabilize
> > the
> > enzyme.
> >
> > Any suggestions for additives that may also stabilize it during
> > functional
> > assays are welcome. I realize that this is potentially a more tricky
> > challenge
> > (the colorimetric assay is limited in terms of pH and salts etc.).
> > Stabilizing it
> > during storage would be a great step.
> >
> > Thank you,
> >
> > Pedro Rocha
> >
> >
>
> ---




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