| Assay Method Information | |
| | Determination of IC50 Values of Kinase Inhibitors |
| Description: | Each plate contained a positive control (1 μM Comp. A for ROCK2 and 1 μM RKI-1447 for ROCK1); a vehicle control (1% DMSO); a blank control (kinase reaction buffer (1×)); a low control (substrate and ATP, without kinase); and an autophosphorylation control (kinase and ATP, without substrate). Test and reference compounds were diluted in 100% DMSO to obtain 10 mM stock solutions. Each compound was tested in 8-serial dilutions in duplicate. The final concentration of DMSO in the reaction was 1% (up to 50 nL). 5 μl/well Kinase Reaction Buffer (1×) was dispensed to blank control wells. 2 μl/well Kinase Reaction Buffer (1×) was dispensed to control without protein (low control) and without substrate (autophosphorylation) wells. The plate was sealed with adhesive film followed by a short spin (1 min, 1000 rpm). 2 μl/well kinase (2.5×) solution was dispensed into relevant wells of the assay plate. The plate was sealed with adhesive film followed by a short spin (1 min, 1000 rpm). 2 μl/well S6K substrate (2.5×) solution was dispensed into relevant wells of the assay plate. The plate was sealed with adhesive film followed by a short spin (1 min, 1000 rpm). 1 μl/well ATP (5×) solution was dispensed into relevant wells of the assay plate. The final volume of reaction was 15 μl. The plate was sealed with adhesive film followed by a short spin (1 min, 1000 rpm). The plate was incubated at 25° C. for 120 minutes with 450 rpm shaking speed. After the incubation was completed the kinase reaction was terminated, residual ATP was depleted from the kinase reaction and ADP was converted to ATP, that is measured in luciferase/luciferin reaction. 5 μl of ADP-Glo™ Reagent was dispensed to stop the kinase reaction and deplete the unconsumed ATP. The plate was sealed with adhesive film followed by a short spin (1 min, 1000 rpm), and was incubated at 25° C. for 40 minutes with 450 rpm shaking speed. |
| Affinity data for this assay | |
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