MRCKα, MRCKβ, ROCK1 and ROCK2 assays are performed using an IMAP fluorescence polarization assay format. 8 to 12 nM of each kinase is incubated for 60 min at room temperature with 100 nM FAM-S6-ribosomal protein derived peptide in the presence of 1 μM ATP and 0.5 mM MgCl2 in 20 mM Tris buffer (pH 7.4) containing 0.01% Tween-20 and 1 mM DTT (MRCKα and β); or 1 μM ATP, 10 mM MgCl2 in 20 mM Tris buffer (pH 7.5) containing 0.25 mM EGTA 0.01% Triton X-100 and 1 mM DTT (ROCK1 and ROCK2). Typically, dose response analysis are performed over concentration ranges from 0.005 to 100 μM. Reactions are stopped by adding 2 assay volumes of 0.25% (v/v) IMAP binding reagent in 1×IMAP binding buffer. After 30 min incubation to allow binding reagent to bind phosphorylated peptide, fluorescence polarization is measured on a plate reader at excitation (470 nm) and emission (530 nm) wavelengths. Inhibition is calculated using no inhibitor and no enzyme controls as 0 and 100% inhibition, respectively. Kinase selectivity profiling is performed by Eurofins with 10 μM ATP and 10 μM BDP5290[2].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.