IUBio

endogenous phosphatase

Paula Murphy paulam at cs.mun.ca
Sat Jul 24 08:40:38 EST 1999


> Also, try a colorimetric method for detection; it is often faster and
>easier to control, with less background
>than the chemiluminescent methods.
> Scott Jamison, Ph.D.
  
Well you see, sensitivity is actually the point of this small experiment.
My supervisor wants me to use chemi. and I am struggling to find a way to
knock out the endogenous AP. I am concurrently using colorimetric methods
but only for comparison sake. No one seems to have commented on my
question about including phosphate in the medium and washing in PBS. Is
this because it is not likely to work?
 
Thanks,
Paula Murphy
> Paula Murphy wrote in message ...
> >I am doing some plaque lifts and subsequently washing a nitrocellulose
> >membrane in 0.5%milk-TBS buffer and secondary antibody conjugated with
> >alkaline phosphatase. The method that I use to detect the bound secondary
> >antibody also detects the endogenous phosphatase. If I add organic
> >phosphate to the medium and wash the membranes with PBS instead of TBS
> >will the production of alkaline phosphatase decrease?(My results are
> >obtained on xray film)
> >
> >--Thanks,
> >paulam at ganymede.cs.mun.ca
> >
> >=========================
> >
> >
> 
> 
> 
> 




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