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ProFluor Tyrosine Phosphatase Assay

by Promega
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  • High-Throughput Detection of Tyrosine Phosphatase Activity

    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 include PTP-1B, CD45, LAR PTPase and YOP-51. 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.

    Protocol Overview

    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 (control for compounds that may inhibit the protease). 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.

    Advantages

    • Minimal Test Compound Interference. The Rhodamine 110 fluorescent signal produced is much higher than the fluorescent signal given off by test compounds.
    • Control Peptide Included (AAF-AMC) for monitoring protease activity, reducing false-positive hits.
    • Simple add-mix-read format reduces the number of plate-handling steps to fewer than that required for other phosphatase assays.
    • No radioactive waste disposal and safety issues.
    • Custom formats available: www.promega.com/custom

  • Protocols

    Complete Protocol

    Download PDF

    ProFluor® Tyrosine Phosphatase Assay Technical Bulletin

    PDF (385 KB)

  • Certificate of Analysis

    Lookup Certificate of Analysis

    Storage Conditions

    -30C TO -10C

    Use Restrictions

    For Research Use Only. Not for Use in Diagnostic Procedures.

  • Resources

    Articles