desperately need help
aa374885 at OAK.CATS.OHIOU.EDU
Mon Mar 10 17:43:30 EST 1997
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
> 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
> - 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 .
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