preventing protein degradation

Dr E. Buxbaum EB15 at
Thu Sep 25 19:04:02 EST 1997

Yankiwski wrote:

> I'm expressing a protein in Sf9 insect cells using a baculovirus vector.
> Upon purification of the 6x his-tagged protein on a nickel affinity column
> and after Western analysis, I get multiple bands, with the majority being
> of smaller size and in greater quantity than the full-length protein.  This
> is the case in both denaturing and nondenaturing purification conditions,
> and is not ameliorated by the use of  "protease inhibitor cocktail"
> tablets, PMSF, and beta-mercaptoethanol in the lysis buffer and in
> subsequent wash and elution steps.  

There is the possibility that your protein is clipped already in the
cells. It may be worthwhile to take some cells up directly in SDS sample
buffer and do a western blott to check for this possibility. 

If the cells realy degrade your protein, you may try to give protease
inhibitors together with the inductor, but in my experience it is
something one has to live with.

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