In article <19970928000100.UAA22816 at ladder01.news.aol.com>, yankiwski at aol.com (Yankiwski) wrote:
#Hi,
#
#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. Any thoughts?
Yes. If the amount of "degradation" (you first have to make sure it's not
crappy antibodies staining just about everything) is the same with intact cell
lysed with loading buffer and in your homogenates, then your protein has
very fast turnover cycle and there is little you can do. Certainly not
inhibitors (PMSF, as membrane-permeable inhibitor, might help, though).
- Dima
