| Assay Method Information | |
| | Enzymatic Assay |
| Description: | The assay is done using an ADP-Glo Kinase Assay Kit (Promega, Catalog #V9102) according to the manufacturer's protocol with the following modifications. Briefly, hASK1 (0.25 nM) and MKK6 (300 nM) in a buffer (10 mM MOPS pH 7.0; 10 mM Mg-Acetate; 1 mM DTT; 0.025% NP-40; 0.05% BSA; 1.5% glycerol) are incubated with ASK1 inhibitors at varying concentrations ranging from 10.00 uM to 0.17 nM for 15 minutes, followed by incubation with ATP (100 uM) for 30 minutes at room temperature. ADP-Glo Reagent is added to terminate the kinase reaction and deplete the remaining ATP. The Kinase Detection Reagent is then added to convert ADP to ATP. The newly synthesized ATP is measured using a luciferase/luciferin reaction, and the luminescence determined by Envision (PerkinElmer). |
| Affinity data for this assay | |
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