ProFluor® Ser/Thr PPase Assay

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 begins with a standard phosphatase reaction performed with a 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 reaction. Following the phosphatase reaction, a termination buffer containing a proteas...

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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 begins with a standard phosphatase reaction performed with a 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 reaction. Following the phosphatase reaction, a termination buffer containing a protease reagent is added, which simultaneously stops the phosphatase reaction and removes amino acids specifically from the nonphosphorylated substrate, liberating highly fluorescent rhodamine 110. Phosphorylated substrate, however, is resistant to digestion by the protease reagent and remains nonfluorescent. Thus, fluorescence intensity measured in this assay is directly correlated with phosphatase activity. 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.

Features - Benefits

• Achieve Highly Predictive Results: Robust Z´ values greater than 0.7 in either 96- or 384-well plate formats.
• Observe Minimal Test Compound Interference: Rhodamine 110 fluorescent signal produced is much higher than the fluorescent signal given off by test compounds.
• Control Peptide Included: Use AAF-AMC control peptide to monitor protease activity and reduce false-positive hits.
• Simplify Your Assays: Add-mix-read format reduces the number of steps.
• Non-Radioactive: No radioactive waste disposal and safety issues.
• Choose Your Configuration: Learn more about our custom options for this product at: www.promega.com/myway/

Applications

For more information, see the Protocols & Applications Guide.

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