Preventing protein deradation

Mr. G. Morley gmorley at hgmp.mrc.ac.uk
Thu Sep 25 04:35:21 EST 1997

>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.

Hi.. not entirely sure as to the capabilities of insect cells in
 producing proteins - but is it possible that what you are seeing
in you gel is not degradation fragments but in fact is the reverse -
ie: glycosylation precursors to your mature protein?
Of course if insect cells can't glycosylate your protein then this
can't be the case...
					Just a thought,
									Gary Morley
								gmorley at rpms.ac.uk

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