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 and reduce false-positive hits.