The ProFluor® PKA Assay measures protein kinase A activity using purified kinase 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 Bisamide Rhodamine 110 peptide substrate (PKA R110 Substrate). In this configuration the substrate is nonfluorescent. Following the kinase reaction, a termination buffer containing a Protease Reagent is added, simultaneously stopping the kinase reaction, removing amino acids specifically from the nonphosphorylated PKA R110 Substrate, and producing highly fluorescent Rhodamine 110. The phosphorylated PKA R110 Substrate is resistant to digestion by the Protease Reagent and remains nonfluorescent. Thus, the fluorescence intensity measured in the assay is inversely correlated with kinase activity.
Minimal Test Compound Interference
The Rhodamine 110 fluorescent signal produced is much higher than the fluorescent signal given off by test compounds. The assay produces excellent Z´-factor values (>0.8) in either 96- or 384-well plate formats and easily distinguishes known PKA inhibitors from other compounds.
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