Microplates are coated with the capture antibody provided in the assay. Samples containing phosphatase are added.
Any phosphatase present is bound by the immobilized antibody. Unbound materials are washed away.
A synthetic phosphopeptide substrate is added that is dephosphorylated by active phosphatase to generate free phosphate and unphosphorylated peptide. The free phosphate is detected by a sensitive dye binding assay using malachite green. By calculating the rate of phosphate release, the activity of the specific phosphatase of interest is determined.