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
Euan



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,
>>reduced).

>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!

>Brian
-- 
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 



More information about the Methods mailing list