Q. regarding stabilizing enzyme (for storage)
dk at no.email.thankstospam.net
Sun Jun 30 20:45:43 EST 2002
Pedro Rocha <p.s.c.rocha at durham.ac.uk> wrote:
>I would appreciate any input from protein experts on how to stabilize a
>protein (obtained after overexpression in E.coli).
>This particular enzyme is purified using Ni-NTA resin, being eluted with
>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.
>-20C. Addition of various %s of glycerol did not help at all. I would
>appreciate any suggestions of additives that could be tried to stabilize
If you can come up with an explanation as to what it is about it that
makes it unstable, the decision might be a lot more rational. Generally
speaking you have plenty of things to try: immediate neutralization
to pH > 7.0, protease inhibitors, sugars (sucrose, trehalose, xylitol),
detegents (triton X-100, chaps, octyl-glucoside), betaine, DTT ...
the list is endless. If it can work for you, perhaps easiest solution
is to "drop-freeze" in LN2 (25 ul drops directly into LN2; you end
up with plenty of beaded protein aliquotes that you can store at -80C).
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