| Assay Method Information | |
| | Assay of In Vitro Kinase Activity |
| Description: | 1. Purpose of the Assay:The ability of compounds to inhibit ERK1 and 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 Assay:a) The substrate MBP was prepared in freshly prepared reaction buffer;b) ERK1 (or ERK2) kinase was added to the above-mentioned MBP solution and mixed gently.c) 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;d)33P-ATP (specific concentration of 10 μCi/μL) was added to the reaction system, and the reaction was started at this time;e) The mixture was incubated at room temperature for 2 hours;f) The amount of radioactivity was detected by filter-binding method;g) ERK1 (or 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). |
| Affinity data for this assay | |
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