| Assay Method Information | |
| | In Vitro Enzyme Activity Assay |
| Description: | The substrate Z′-LYTE Ser/Thr 3 Peptide, the phosphorylation substrate Z′-LYTE Ser/Thr 3 Phospho-peptide, 1× kinase buffer (5× kinase buffer was diluted with ultra pure water by five times), ATP, Development Reagent A, Development Buffer, Stop Reagent were balanced to room temperature for use. The screening concentrations for detecting the effect of the compounds of the invention on the ERK kinase activity were seven 3-fold serial dilutions starting from 1 μM (from 0.2 μM for the positive drug control) using 4% DMSO as co-solvent. 5 μL enzyme system (50 mM HEPES pH 7.5, 1 mM EGTA, 10 mM MgCl2, 0.01% Brij-35, 4 uM substrate, 0.8 ng/μL enzyme), 2.5 μL Compound, 2.5 μL 400 μM ATP were added into 384 well microplate, followed by incubation at room temperature for 60 min in the dark. After the reaction was completed, 5 μl the developing agent (Development Reagent A) diluted with the developing buffer (Development Buffer) was added to all reaction wells, followed by incubation at the room temperature for 60 min in the dark. 5 μL the terminating agent was added to each well to terminate the reaction. The fluorescence was determined using the multil-mode microplate reader PerkinElmer EnVision (excitation wavelength 400 nm, emission wavelength 460 nm and 528 nm). |
| Affinity data for this assay | |
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