desperately need help

[Keld Sorensen]

AAAACc wrote:
> 
> Hi people who are expert for western!
> 
> I am doing western using PY20 (antiphosphotyrosine antibody) and
> developing with ECL  but it did not work. I checked every condition it is
> still not working.
> 
>  Any suggestion is appreciated.
> 
> Thank you

Did you use milk as the blocking reagent? Milk has phosphoproteins and
could give you a problem. You have to give specifics though - right now
nobody
can help you - it would be like bringing your car in for repair and say:
"My car is not working right, please fix it!" - 
So please explain the details of your WB along with observations
(symptoms)
- details such as:
how much sample
how run
how transferred
transferred to what membrane
what conc. of PY20 
what enz-ab conjugate or are you suing PY-20 enzyme conjugate
What substrate - etc etc etc.