How can I test if my alkaline phosphatase enzyme is working or not?

Question

Would a substrate run differently on a gel electrophoresis if the alkaline phosphatase worked on it (it's linear double stranded DNA). I'm having an experiment (ligation) fail repeatedly and I want to determine if the alkaline phosphatase is inactive and that's why it's failing.

Answer 1

p-Nitrophenyl Phosphate (PNPP) is a non-proteinaceous, non-specific substrate used to assay protein, alkaline and acid phosphatases. The PNPP phosphatase activity is measured using a continuous or single-point spectrophotometric assay based on the ability of phosphatases to catalyze the hydrolysis of PNPP to p-nitrophenol, a chromogenic product with absorbance at 405 nm.

Assay Conditions:

The reaction mixture contains 100mM glycine, pH 10.4, 1mM MgCl2, 1mM ZnCl2, 10mM p-nitrophenyl phosphate and 0.001-0.1 units  Alkaline Phosphatase (AP). The change in absorbance at 405 nm is monitored (3050 µl reaction volume).

http://www.getsapfacts.com/aboutSAP.asp