ProFluor® Src-Family Kinase Assay

The ProFluor® Src-Family Kinase Assay measures the activity of purified Src-family tyrosine kinases (Src, Lck, Lyn, Fyn, and Hck tested) in a multiwell plate format and involves "add-mix-read" steps only—ideal for high-throughput applications. The assay begins with a standard kinase reaction performed with a provided Src-family kinase bisamide rhodamine 110 peptide substrate. Following the kinase reaction, a termination buffer containing a protease reagent is added, which simultaneously stops the kinase reaction and removes amino acids specifically from the nonphosphorylated substrate, liberating highly fluorescen...

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The ProFluor® Src-Family Kinase Assay measures the activity of purified Src-family tyrosine kinases (Src, Lck, Lyn, Fyn, and Hck tested) in a multiwell plate format and involves "add-mix-read" steps only—ideal for high-throughput applications. The assay begins with a standard kinase reaction performed with a provided Src-family kinase bisamide rhodamine 110 peptide substrate. Following the kinase reaction, a termination buffer containing a protease reagent is added, which simultaneously stops the kinase 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 inversely correlated with kinase activity. A control peptide (AAF-AMC) is included to control for compounds that may inhibit the protease. The assay produces excellent Z´ values (>0.7) in either 96- or 384-well plate formats and easily distinguishes known Src-family kinase 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.
• Homogeneous: Add-mix-read format reduces the number of plate-handling steps.
• Non-Radioactive: No radioactive waste disposal and safety issues.

Applications

• HTS Src-Family kinase screening.

For more information, see the Protocols & Applications Guide.

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