ProFluor® Ser/Thr PPase Assay Technical Bulletin

Literature # TB324

The ProFluor® Ser/Thr PPase Assay measures purified serine/threonine protein phosphatase activity in a multiwell plate format and involves “add, mix and read” steps only. The assay works with protein phosphatase 1 (PP1), PP2A, PP2B, and PP2C. The assay begins with a standard phosphatase reaction performed in the reaction buffer with the provided phosphorylated bisamide rhodamine 110 peptide substrate (S/T PPase R110 Substrate) and Control AMC Substrate that serves as a control for compounds that may inhibit the protease. In this configuration, both the S/T PPase R110 Substrate and Control AMC Substrate are nonfluorescent. Following the phosphatase reaction, adding a protease solution simultaneously stops the phosphatase reaction and completely digests the dephosphorylated S/T PPase R110 Substrate and Control AMC Substrate, producing highly fluorescent rhodamine 110 and AMC. Phosphorylated S/T PPase R110 Substrate, however, is resistant to protease digestion and remains nonfluorescent. Thus, the R110 fluorescence intensity measured in the assay correlates with phosphatase activity in the presence of active protease, and the AMC fluorescence intensity is an indication of protease activity.

The assay produces Z´ values greater than 0.8 in both 96- and 384-well plate formats and produces IC₅₀ values for known inhibitors that are comparable to those currently reported in the literature. The amount of phosphatase used per well is low (ng/well), and the fluorescent signal is stable (approximately 10 percent change of fluorescence intensity in 4 hours), allowing batch-plate reading.