p-tyr blotting question

Euan R. Taylor etaylor at ccu.umanitoba.ca
Wed Apr 28 10:39:00 EST 1993

Try titering your primary and secondary antibodies against your target
lysate and a sample of IgG run alongside it, keep increasing the dilution
factor until you can see reaction to the lysate, and not to the IgG.
Even monoclonals cross react .
Harlow and Lane (Antibodies, Alaboratory manual) have a good deal of
infromation on this type of thing (though not especially P-Tyr if I
remember rightly)
hope this helps

In <1rjrc5INN6pf at lynx.unm.edu> bhjelle at carina.unm.edu writes:

>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!

Usual disclaimers. These are my own opinions and probably nobody else's.
etaylor at ccu.umanitoba.ca
"All great truths begin life as blasphemies"  George Bernard Shaw 

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