preventing protein degradation

Yankiwski yankiwski at aol.com
Wed Sep 24 21:08:00 EST 1997


Hi,

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.  Assuming specificity of the Ab, and
that the multiple bands are in fact due to degradation, what can be done to
remedy the problem?

Thanks for any suggestions.
Victor



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