Science Catalog 2012 Life Cell Signaling ProFluor® Ser/Thr PPase Assay Product ProFluor® Ser/Thr PPase Assay For Research Use Only. Not for Use in Diagnostic Procedures. Description: 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 both stops the phosphatase reaction and removes amino acids specifically from the nonphosphorylated substrate, liberating highly fluorescent rhodamine 110. However, 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: • Achieve Highly Predictive Results: Robust Z´ values greater than 0.7 in either 96- or 384-well plate formats. • Minimal Test Compound Interference: Fluorescent signal from Rhodamine 110 is much greater than signals from 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 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/ Storage Conditions: Store the entire system at –20°C. Protect the S/T PPase R110 Substrate and Control AMC Substrate from light. Protocol ProFluor® Ser/Thr PPase Assay Technical Bulletin No Phosphatase PP PP PP Protease Reagent Nonfluorescent 20,000 40,000 60,000 80,000 100,000 120,000 140,000 160,000 0 Protease Reagent S/T Phosphatase PP Part# TB324 Size Cat.# Price (Fr) 4 plate V1260 1304.00 8 plate V1261 2348.00 ProFluor® Tyrosine Phosphatase Assay Product ProFluor® Tyrosine Phosphatase Assay For Research Use Only. Not for Use in Diagnostic Procedures. Description: The ProFluor® Tyrosine Phosphatase Assay measures purified tyrosine phosphatase enzyme activity in a multiwell plate format and involves “add-mix-read” steps only—ideal for high-throughput applications. Tyrosine phosphatases tested with the assay include PTP-1B, CD45, LAR PTPase and YOP-51. The assay begins with a standard phosphatase reaction performed with a provided phosphorylated bisamide rhodamine 110 peptide substrate (PTPase R110 Substrate) and Control AMC Substrate that serves as a control for compounds that may inhibit the protease. 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 (>0.7) in either 96- or 384-well plate formats and easily distinguishes known phosphatase inhibitors from other compounds. Features: • Achieve Highly Predictive Results: Robust Z´ values greater than 0.8 in either 96- or 384-well plate formats. • Observe Minimal Test Compound Interference: Substrate used at micromolar concentration. Rhodamine 110 fluorescent signal produced is much greater than the fluorescent signal given off by test compounds. • Control Peptide Included: Control peptide (AAF-AMC) included that is used to monitor protease activity. Reduces false positive hits. • Simplify Your Assays: Simple add-mix-read format reduces the number of plate-handling steps to fewer than that required for other phosphatase assays. • Save Time: Minimal throughput time compared to the multiple steps and lengthy incubations with other phoshatase assays. • 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/ Storage Conditions: Store the entire system at –20°C. Protect the PTPase R110 Substrate and Control AMC Substrate from light. Protocol ProFluor® Tyrosine Phosphatase Assay Technical Bulletin Part# TB334 Size Cat.# Price (Fr) 4 plate V1280 1304.00 8 plate V1281 2348.00 Fluorescent r2 = 0.99 0102030405060708090 100 Percent Nonphosphorylated Peptide Effect of phosphopeptide content on fluorescence intensity. The graph shows the average RFU obtained after a 90-minute digestion of mixtures of nonphosphorylated S/T PPase R110 Substrate and phosphorylated substrate as indicated to mimic a phosphatase titration. 70 For complete and up-to-date product information visit: www.promega.com/catalog RFU 4166MB Pagina 73
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