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Abstract for ProFluor® Src-Family Kinase Assay
The ProFluor® Src-Family Kinase Assay measures the enzymatic activity
of the 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.
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