ProFluor® Src-Family Kinase Assay Technical Bulletin

Literature # TB331

The ProFluor® Src-Family Kinase Assay measures the enzymatic activity of Src-family tyrosine kinases such as Src, Lck, Fyn, Lyn and Hck using purified enzymes in a multiwell-plate format and involves "add, mix and read" steps only. The assay begins with a standard kinase reaction with the provided rhodamine 110 peptide substrate (Src-Family Kinase R110 Substrate). Following the kinase reaction, addition of the Termination Buffer, which contains a Protease Reagent, simultaneously stops the kinase reaction and removes amino acids from the nonphosphorylated substrate to generate the highly fluorescent rhodamine 110. The phosphorylated substrate is resistant to digestion by the Protease Reagent and remains nonfluorescent. Thus, the measured fluorescence intensity in the assay is inversely correlated with kinase activity. The fluorescent signal is very stable (<15% change of fluorescence intensity over 4 hours), allowing batch plate reading. The assay produces Z′-factor values greater than 0.7 in either 96-well or 384-well plate formats and easily identifies known kinase inhibitors in a screen of library compounds. The assay produces IC₅₀ values for known inhibitors that are comparable to those reported in literature.