desperately need help

[AAAACc]

First of all, I will thank you attention my problem,

On Mon, 10 Mar 1997, Keld Sorensen wrote:

> 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

I am not using milk as blocking reagent, I am using 4 % BSA. 

> 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 
I am using 25-40 microliter cell lysate

> how run

I am running 100 volt for 1 hour

> how transferred

I think transfer is okey since I am using colored marker and you can see
amrker on the membrane
  
> transferred to what membrane

hybond cellulose membrane
> what conc. of PY20 

I am using 2 miroliter in 25 ml blocking solution.

> what enz-ab conjugate or are you suing PY-20 enzyme conjugate

I am using PY20-HRP conjugate

> What substrate - etc etc etc.

I am using ECL from amersham.> 
> 


Thank you .