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Review date: 18 May 2009 | ProFluor® PKA Assay
The ProFluor® PKA Assay(a) 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 PKA 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 PKA substrate, liberating highly fluorescent rhodamine 110. Phosphorylated PKA 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. 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.