| Assay Method Information | |
| | Biological Assay |
| Description: | Experimental example 1: EGFR, ITK, TEC, and BTK kinase test.1. Reaction condition:Buffer condition: 20 mM HEPES(pH 7.5), 10 mM MgCl2, 1 mM EGTA, 0.02% Brij35, 0.02 mg/mL BSA, 0.1 mm Na3VO4, 2 mm; DTT, 1% DMSO.2. Reaction procedure:2.1. An indicator substrate was prepared in a reaction buffer prepared newly.2.2. A cofactor required was transferred to the substrate solution above.2.3. A kinase indicated was injected into the substrate solution and mixed gently.2.4. An acoustic technology was used to convey a compound in DMSO to a kinase reaction mixture (Echo550).2.5. 33P-ATP (specific activity 0.01 μci/μL, finally) was fed into the reaction mixture to initiate a reaction. (ATP final concentrations were 2 μM, 5 μM, and 5 μM respectively).2.6. The kinase reaction was incubated at room temperature for 120 min.2.7. The reaction was recorded on P81 ion exchange paper (Whatman #3698-915).2.8. 0.75% phosphoric acid was widely used to clean a filter.2.9. The remaining radioactive phosphorylation substrate on filter paper was measured.3. Data analysis: the kinase activity data was expressed as a percentage of the remaining kinase activity in the test sample compared to the carrier (dimethyl sulfoxide) reaction. The IC50 value and curve fitting were obtained by using Prism4 software (GraphPad). |
| Affinity data for this assay | |
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