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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