p-tyr blotting question

bhjelle at carina.unm.edu bhjelle at carina.unm.edu
Tue Apr 27 12:43:33 EST 1993

In article <Pine.3.05.9304221517.A26216-b100000 at post.its.mcw.edu> fgarbrec at POST.ITS.MCW.EDU (Frederick Garbrecht) writes:
>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,

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

You might try preincubating your primary and/or secondary
antibody in the presence of a large excess of mouse serum,
so that whatever nonspecific interaction you are observing
between your antibodies and the murine antibodies is
removed in the liquid phase before touching your membrane.
I would suggest preincubation in 1-5% mouse serum for
4h at RT.

No guarantees!


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