Yankiwski (yankiwski at aol.com) wrote:
> Thanks to everyone for the suggestions I've gotten. To answer some
> questions, the protein I'm expressing is 6x-His tagged at the N-terminus,
> and the Ab I'm using is a polyclonal against the protein rather than the
> tag itself. Silver stained gels shows many more bands than my Westerns do.
> Purification of the protein on two different resins, one Ni-NTA and the
> other Talon, produced similar multi-banded results. It is possible that
> the protein is expressed in a cell-cycle dependent fashion so it may be
> degraded as the cells pass through certain stages of the cycle. If this is
> the case, I suppose that using any combination of protease inhibitors will
> be futile.
I have been told that Sf9 cells don't do a lot of cell cycling after
infection with baculovirus. You could try to play around with the
infection time, maybe cultivate at lower temperatures or compare
expression in monolayers vs. suspension culture. As Dima suggested,
it may indeed be possible to add PMSF shortly after infection since
your cells die anyway.
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