reversible protein staining
klenchin at facstaff.REMOVE_TO_REPLY.wisc.edu
Fri Dec 1 16:12:00 EST 2000
marese at ircc.unito.it (Marco Arese) wrote:
:Since I'm having huge troubles purifiyng my His-tagged protein with
:conventional metal affinity, I think that my last chance is to run my
:sample on a native acrylamide gel, stain it with some staining like
:zinc, which should be reversible, cut the band corresponding to my
:protein and recover it in some ways. Did anyone ever tried that? In
:particular did anybody recover an intact protein from a gel after a
:reversible staining? How big an effort would be to obtain microgram
:amounts of protein?
IMHO, the whole procedure is not worth it for microgram amounts
of the recombinant protein (but I think it should work; most likely
you will end up with pretty dirty protein when run on SDS gel;
native gels rarely give good resolution of complex mixtures.
What's wrong with IMAC? Poor expression? Does not bind at
all? "My" protein binds very poorly and to elute even at 10 mM
imidazole. No problems - I just load without any, wash extensively
with 5 mM, briefly with 10 mM, elute with bump of 100 mM
and after additional ammonium sulphate pption the protein
is about 95% pure (but that is reasonaly good expression levels
in insect cells).
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