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Promega Corporation

ProFluor® Tyrosine Phosphatase Assay Technical Bulletin

Literature # TB334

The ProFluor® Tyrosine Phosphatase Assay measures the enzyme activity of tyrosine phosphatases using purified enzymes in a multiwell-plate format and involves “add, mix and read” steps only. The assay begins with a standard phosphatase reaction performed in the provided reaction buffer, which contains a bisamide rhodamine 110 phosphopeptide substrate (PTPase R110 Substrate) and a Control AMC Substrate that serves as a control for compounds that may inhibit the protease. In this configuration, both the PTPase R110 Substrate and Control AMC Substrate are nonfluorescent. Following the phosphatase reaction, addition of a protease solution simultaneously stops the phosphatase reaction and completely digests the nonphosphorylated PTPase R110 Substrate and Control AMC substrate, producing highly fluorescent rhodamine 110 and AMC. The phosphorylated substrate, however, is resistant to digestion by the Protease Reagent and remains nonfluorescent. Thus, the measured fluorescence intensity in the assay correlates with phosphatase activity. The fluorescent signal is very stable (<20% change of fluorescence intensity over 4 hours), allowing batch-plate reading. The assay produces Z´-factor values greater than 0.7 in either 96-well or 384-well plate formats, and it identifies known phosphatase inhibitors and can be used to identify inhibitors in a screen of library compounds. The assay produces IC50 values for known inhibitors that are comparable to those reported in literature.

Printed in USA. Revised 6/09.

 

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