IEF Western?

Robert Means remeans at fas.harvard.edu
Wed Aug 26 12:11:59 EST 1998


Hello Everyone,
	I haven't done IEF before so I'm hunting around in the dark with
all aspects of this experiment. At any point, my protein is a little
large, 120kD. I'm treating it with different glycosidases and I'm not able
to resolve the small changes in MW that are occuring using SDS-PAGE. One
thing that I thought of doing was looking at charge change after
neuraminidase treatment. My question is, can I somehow transfer the
proteins to a membrane for antibody staining after IEF? The proteins will
have a net neutral charge (if I can make the IEF work) so they won't
transfer by  normal blotting, right? Can I soak the ampholytes out and
somehow impart a charge on the protein and then blot? My samples have too
many proteins to detect my protein of interest just by staining the gel.
Any advice would be great. Also, if somebody has a better way of resolving
2 or 3kD shifts in a 120kD protein I would like to hear about it.

Thanks,
Bob Means
NERPRC/Harvard University
remeans at husc.harvard.edu




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