>>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,
gmorley at rpms.ac.uk