Z’-LYTE in vitro kinase assay

The Z’Lyte in vitro kinase assay (Life Technologies) is described in detail elsewhere [26,27]. Assays were performed in quadruplicate in 384-well low volume, non-binding, black polystyrene microplates (Corning) according to the manufacturer’s instructions. Briefly, this assay measures phosphorylation of the Tyr2 peptide substrate which is tagged with coumarin and fluorescein on its N- and C-termini, respectively, to form a FRET pair. After the kinase reaction, a development step involves site-specific proteolytic cleavage of the unphosphorylated but not the phosphorylated peptide. Peptide cleavage results in loss of the FRET signal. Kinase reactions were run in 50 mM HEPES, pH 7.5, 10 mM MgCl₂, 1 mM EGTA, and 0.01% BRIJ-35 (buffer supplied by the manufacturer). Kinase reactions were initiated by the addition of ATP at the K_m for each kinase and Tyr2 peptide substrate (1 μM). Following incubation for 1 h at ambient temperature, reactions were quenched by addition of the development protease, and coumarin and fluorescein fluorescence were measured 1 h later on a Molecular Devices SpectraMax M5 plate reader. Data are expressed as a ratio of the coumarin (445 nm) to fluorescein FRET (520 nM) emissions normalized to signals observed in the absence of ATP (negative control) and with a positive control peptide in the absence of kinase (Tyr2 that is 100% phosphorylated). For experiments with inhibitors, compounds were preincubated with kinase for 30 min prior to initiation of the reaction by the addition of ATP.