The ProFluor® Ser/Thr PPase Assay measures purified serine/threonine protein phosphatase activity in a multiwell plate format and involves "add-mix-read" steps only—ideal for high-throughput applications. The assay works with protein phosphatase 1 (PP1), PP2A, PP2B and PP2C. The assay produces excellent Z´ values (>0.8) in either 96- or 384-well plate formats and easily distinguishes known phosphatase inhibitors from other compounds.
The assay begins with a standard phosphatase reaction performed with the provided phosphorylated bisamide rhodamine 110 peptide substrate (S/T PPase R110 Substrate) and Control AMC Substrate (control for compounds that may inhibit the protease reaction). Following the phosphatase reaction, a termination buffer containing a protease reagent is added, which stops the phosphatase reaction and removes amino acids specifically from the nonphosphorylated substrate, liberating highly fluorescent rhodamine 110. Phosphorylated substrate is resistant to digestion by the protease reagent and remains nonfluorescent. Thus, fluorescence intensity measured in this assay is directly correlated with phosphatase activity.