SPAGNOL at PLAUTO.CSATA.IT wrote:
: I cloned a glycoprotein in a cell line and have antibodies against its
: sugar moiety. I do immunofluorescence and the protein is there, then I
: perform an immunoblot with the same Ab and nothing stains. I arrived to load
: exctracts of 10,000,000 cell on SDS PAGE- still nothing. I strongly confide
: in the expertiseof of somebody of you out there, dear colleagues.
: Sincerely
: Giorgio
I dont think it is particualarly unusual for antibodies to work in one
test and not another. Flow cytometry and western blotting are two very
different environments and the antigen may be in very different
configurations (even in non-denaturing gels). Monoclonal Abs are
especially susceptable to these difficulties. The amount of antigen on
the blot is probably not the problem. Why not try a different test, like
cell-elisa or immuno-gold labelling?
Greg
