| Assay Method Information | |
| | Kinase Assay |
| Description: | The inhibitory activity of some compounds of the present disclosure against FGFR1, FGFR2, FGFR3, FGFR4, KDR was tested by mobility shift assay (concentration of ATP is Km value).Test method:Reagent: basic kinase buffer: 50 mM HEPES (pH 7.5); 0.0015% Brij-35Stop buffer: 100 mM HEPES (pH 7.5); 0.015% Brij-35; 0.2% Coating Reagent #3; 50 mM EDTAPreparing of compounds: dilute test compounds to specific concentration using 100% DMSOReaction process: 1) preparing of 2.5× enzyme solution adding kinase to 1× basic kinase buffer 2) preparing of 2.5× peptide solution adding FAM-labeled peptide and ATP to 1× basic kinase buffer 3) preparing of analysis board transferring 10 μL of test compound to 384-well plate, adding 904, of 1× basic kinase buffer 4) adding 104, of 2.5× enzyme solution to each well of the assay plate and incubating for 10 min at room temperature 5) adding 104, of 2.5× enzyme solution to each well of the assay plate and incubating for specific time at 28° C. 6) stop the reaction by adding 254, of stop solution to each well 7) reading data with Caliper and calculating IC50 value. |
| Affinity data for this assay | |
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