4.5. IC50 Assay

Initially, the tested compound was dissolved in DMSO to make a 10 mM stock solution, and the stock solution was then diluted to a drug solution with 50× test concentrations for later use, wherein the test concentrations were reached through dilution at a 10-fold gradient and were 0.1 nM, 1 nM, 10 nM, 100 nM, 1000 nM, 10,000 nM, respectively. Firstly, 2× ATP and substrate solution and 2× kinase and metal solution was prepared using assay buffer (MgCl2 2 mM, MnCl2 1 mM, SEB 12.5 nM, DTT 0.5 mM). From each well of the 96-well plate, 25 μL of the drug solution was taken and then transferred to a 384-well plate which was provided with 2 duplicate wells. Then, 2.5 μL of 2× kinase and metal solution was mixed and incubated in a polystyrene-coated 384-assay plate for 10 min at 25 °C. Then, 2× XL665 and antibody solution was prepared with detection buffer. An amount of 5 μL of Kinase Detection Reagent was added to the well, and incubated for 60 min at 25 °C. The fluorescence signals of 620 nm (Cryptate) and 665 nm (XL665) were read by a microtiter-plate reader. At last, the IC₅₀ value of JNK3 kinase was calculated with the following equations.