| Assay Method Information | |
| | In Vitro Biochemical Kinase Assay of FGFR 1-3 |
| Description: | 1. The kinase reaction in the present invention was carried out in 384-well plates, the kinase(Carna) at a certain concentration and ATP at a certain concentration and 1 μM of peptide FAM-P22 (GL Biochem, Cat. No. 112393)) was incubated to react for a certain time at 28° C. in a reaction system consisting of 50 mM HEPES, pH47.5, 0.0015% Brij-35 and basic kinase buffer; for FGFR1, the enzyme concentration was 0.25 nM and ATP concentration was 382 μM and the reaction time was 20 minutes; for FGFR2, the enzyme concentration was 2.5 nM, ATP concentration was 1 μM, and the reaction time was 40 minutes; for FGFR3, the enzyme concentration was 8 nM, ATP concentration was 4.7 μM, and the reaction time was 30 minutes:2. The reaction was terminated with a stop solution (100 mM HEPES, pH 7.5, 0.2% Caliper coating, reagent, 50 mM EDTA and 0.015% Brij35);3. The plate with the terminated kinase reaction was transferred to the Caliper workstation to read the data;4. the phosphorylated and unphosphorylated peptides were separated by the Caliper microfluid migration shift technique, and the analyte was transferred by a constant buffer flow through the chip, the migration of the substrate peptide was monitored by the labeled fluorescent signal, and the kinase activity was calculated by the amount of the phosphate-based peptide formed.5. IC50 was determined by non-linear regression analysis of percent inhibition at different concentration level of the compound. |
| Affinity data for this assay | |
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