Paula:
You might try 2 things. First, bake the membrane before adding antibodies.
If your organism has a heat-sensitive phosphatase, this will help inactivate
the endogenous. Also, try different membrane(s). Some membranes give a
higher background level than others. 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.
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>>=========================
>>
