CHEMBL3706000

Inhibition Assay: In a final reaction volume of 25 ul, ROCK-II (h) (5-10 mU) is incubated with 50 mM Tris pH 7.5, 0.1 mM EGTA, 30 uM KEAKEKRQEQIAKRRRLSSLRASTSKSGGSQK (SEQ ID NO:1), 10 mM MgAcetate and [gamma-33P-ATP] (specific activity approx. 500 cpm/pmol, concentration as required). The reaction is initiated by the addition of the MgATP mix. After incubation for 40 minutes at room temperature, the reaction is stopped by the addition of 5 ul of a 3% phosphoric acid solution. 10 ul of the reaction is then spotted onto a P30 filtermat and washed three times for 5 minutes in 75 mM phosphoric acid and once in methanol prior to drying and scintillation counting.