| Assay Method Information | |
| | Assay of In Vitro Kinase Activity |
| Description: | 1. Purpose of the Assay:The ability of compounds to inhibit ERK2 kinase activity was measured.2. Assay Buffer:20 mM Hepes (pH 7.5), 10 mM MgCl2, 1 mM ethylenebis(oxyethylenenitrilo)tetraacetic acid (EGTA), 0.02% Brij35, 0.02 mg/mL bovine serum albumin (BSA), 0.1 mM Na3VO4, 2 mM dithiothreitol (DTT), 1% DMSO.3. Processing of Compound:The assay compound was dissolved in 100% DMSO to prepare a stock solution of specific concentration. The compound was serially diluted in DMSO solution using Integra Viaflo Assist smart pipette.4. Method of the Assay1) The substrate MBP was prepared in freshly prepared reaction buffer;2) ERK2 kinase was added to the above-mentioned MBP solution and mixed gently;3) The compound dissolved in 100% DMSO was added to the kinase reaction system using ultrasound technology (Echo550; nanoliter range), and the mixture was incubated at room temperature for 20 minutes;4) 33P-ATP (specific concentration of 10 μCi/μL) was added to the reaction system, and the reaction was started at this time;5) The mixture was incubated at room temperature for 2 hours;6) The amount of radioactivity was detected by filter-binding method;7) ERK2 kinase activity was calculated as the ratio of the remaining kinase activity in the assay sample to the kinase activity of the control group (treated by DMSO). Curve was fitted using Prism (GraphPad software) and IC50 values were calculated.5. The Assay Results were Shown in Table 2 |
| Affinity data for this assay | |
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