Q. regarding stabilizing enzyme (for storage)

Anthea Scothern anthea.scothern at umist.ac.uk
Mon Jul 1 03:43:19 EST 2002


"Pedro Rocha" <p.s.c.rocha at durham.ac.uk> wrote in message
news:3D1F7020.BEC88885 at durham.ac.uk...
> Hello,
>
> I would appreciate any input from protein experts on how to stabilize a
> purified
> protein (obtained after 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.
>

How pure is your protein?  Is the activity declining or is it degrading?  I
usually find I need more than one step of purification to get clean protein.

I routinely add 0.5mM 2-beta mercaptoethanol to my purification buffers to
retain the activity of my proteins - this is a low enough concentration to
avoid reducing the nickel on the resin.  After completion of the
purification, I use 5mM for storage at -80C in 50 - 100ul aliquots.

I am purifying protein for functional assays and specifically have to avoid
the use of glycerol and detergents, as they can interfere with these
experiments.

Watch out with the mercapto - it's toxic as hell.  You could try DTT as
well, but not for your nickel column as it will reduce the metal.

HTH,
Anthea.


--
Anthea Scothern
Post-transcriptional Control Group
Department of Biomolecular Science
UMIST
PO Box 88
Manchester M60 1QD







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