Endogenous phosphatase

C Jamison jamisoncsNO at SPAMaccessam.com
Sat Jul 24 07:12:10 EST 1999


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)
>paulam at ganymede.cs.mun.ca

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