ProFluor® PKA Assay Technical Bulletin

Literature # TB315

The ProFluor® PKA Assay measures protein kinase A activity using purified kinase in a multiwell plate format and involves "add, mix, and read" steps only. The assay begins with a standard kinase reaction performed in the provided reaction buffer with a provided Bisamide Rhodamine 110 peptide substrate (PKA R110 Substrate). In this configuration the PKA R110 Substrate is nonfluorescent. Following the kinase reaction, addition of a termination buffer, which contains a Protease Reagent, simultaneously stops the kinase reaction and removes amino acids specifically from the nonphosphorylated PKA R110 Substrate, 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.