Anti-phosphotyrosine immunoblot

[John Hines]

Hi all,

I am almost at the end of my chain here with this antiphosphotyrosine
immunoblotting.  Originally, I was using the PY-20 antibody and Amersham
ECL detection, but my blots always came out really blotchy, although I
usually got some bands.  So I switched to colorimetric detection (Promega
ProtoBlot HRP and AP), and now I am getting no bands at all.  Does PY-20
go bad?  Does anyone have any tips to make the ECL work any better?  I
know there is a story in my study, but I simply can't get a decent blot to
convince other people!  Am I the only one with such problems with ECL??

Argh!

John