Blocking of PVDF membrane when using anti-phosphotyrosine Antibody

Mikkel mikpedNOmiSPAM at rh.dk.invalid
Tue Mar 14 08:20:35 EST 2000


I have a problem regarding blocking of PVDF membranes when
detecting phosphorylated proteins with antiphosphotyrosine
antibodies (PY-20).
For detection I use AP conjugated Rabit anti mouse IgG and
the NBT/BCIP tablets (Boehringer Mannheim) for development.
The product specifications sheet states that 1 % BSA, 10 mM
Tris pH 7.5, 100 mM NaCL, 0.1 % Tween should be used for
blocking.
However every time i develop the blot the whole membrane
turns purple/brown.
Can anybody please give me an advice regarding blocking of
the PVDF membrane when using PY-20.


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