p-tyr blotting question

Frederick Garbrecht fgarbrec at POST.ITS.MCW.EDU
Thu Apr 22 15:07:17 EST 1993


I have been fooling around trying to do phosphotyrosine western blotting
using human PBMC that have been stimulated with various murine monoclonal
antibodies.  In some of the blots, there are bands that look suspiciously
like heavy and light chain immunoglobulin bands (50,000 and 20,000 kD,
reduced).  The primary antibody that I use is the polyclonal rabbit
anti-p-tyr from UBI, and the secondary is a goat-anti-rabbit-IgG-HRP from
BioRad.  I develop the blots using the ECL biolum kit.  I would have
thought that the murine antibodies that are present in the lysate (from
the cell stimulation) would be denatured by the SDS in the
electrophoresis/blotting, and would not be detected by the secondary
antibody therefore (especially since the secondary antibody is an
anti-rabbit, not an anti-mouse!).  Does anybody have any experiance with
this kind of system; any helpful hints (including specific reagents) would
be appreciated.  (By the way, I have no connection with BioRad, UBI,
Amersham, except that I help keep them all in business by spending too
much money on their stuff).

Thanks,

Pulling my hair out-
Fred Garbrecht
Medical College of Wisconsin
fgarbrec at post.its.mcw.edu




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