| Assay Method Information | |
| | In Vitro Enzymatic Inhibitory Activity |
| Description: | Reagents:Basic reaction buffer: 20 mM hydroxyethylpiperazine ethane sulfonic acid (pH 7.5), 10 mM magnesium chloride, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL bovine serum albumin, 0.1 mMNa3VO4, 2 mM DTT, 1% DMSONecessary cofactors were separately added to the CSF-1R kinase reaction.Enzyme: CSF-1R, 2.5 nMTreatment:The test compounds were prepared into solutions of specified concentrations in 100% DMSO, and the solutions were serially diluted in DMSO using the Integra Viaflo Assist.Procedures:1. A fresh medium preparation reaction buffer was prepared;2. All necessary cofactors were added to the reaction buffer described above;3. The kinase was added into the medium solution and the mixture was shaked gently;4. Solutions of the compounds in DMSO were added to the kinase reaction mixture using an acoustic technique (Echo550; in nanoliter range), and the system was incubated at room temperature for 20 min;5. 33P-ATP (specific activity: 10 μCi/μL) was added to the reaction mixture to initiate the reaction;6. The mixture was incubated for 2 h at room temperature;7. The kinase activity was detected by a filter-binding method;8. The kinase activity IC50 values and the curves were obtained by comparison with the other kinases and the vehicle (DMSO) group using Prism (GraphPad software). |
| Affinity data for this assay | |
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