problem with immunoaffinity column

baudouin baudouin at cix.cict.fr
Tue Oct 10 05:42:44 EST 1995


Hi netters,
I would like to study the composition of a multimeric complex. The
strategy I'd plan to use is separating my complex from crude MonoQ
fraction using antibodies linked to Affigel Hz -which is supposed to
provide the best recognition according to the fact that IgG are
orientated-. My problem is that during the elution (pre-elution buffer
: Tris 0,01 M pH 7,5 ; elution buffer : glycine 0,1 M pH 2,5) I have a
leak of both light and heavy chains of immunoglobulin even after a
pre-run to get rid of non covalently linked IgG. So, the question is
how to get rid of the leak. I've carried the IgG fixation as told in
the instruction manual ; I've also tried mild elution conditions but I
wasn't able in this case to get back the protein.
Thank you for suggestions
Emmanuel



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