| Assay Method Information | |
| | In Vitro Enzymatic Inhibitory Activity Assay |
| Description: | SupplierReagent HEPES Life TechnologiesBRIJ 35 detergent (10%) MerckMgCl2 SigmaHTRF KinEASE-TK kit CisbioFAK SignalchemATP PromegaDTT (DL-Dithiothreitol) SigmaConsumablesTopseal A Perkin Elmer96 Well Plates NuncProxiPlate-384 Plus Perkin ElmerInstrumentsPlate reader Perkin ElmerCentrifuge Eppendorf2. Experimental PROCEDURE1) To 384-well dilution plate was added 50 μL of compound.2) Samples in each column were serially diluted with DMSO at a ratio of 1:3, with 10 samples diluted each time, as well as a control containing only DMSO.3) 0.1 μL of the diluted solution of the compound in each row was transferred to a 384-well assay plate, with 2 replicates for each column.4) To the assay plate was added 5 μL of 2× enzyme solution, centrifuged at 1000 rpm for 1 minute, and incubated at 25° C. for 15 minutes.5) To a 384-well assay plate was added 5 μL of 2× substrate solution.6) Incubation at 25° C. for 60 minutes.7) To assay plate were added 5 μL of Sa-XL665 solution and 5 μL of TK antibody-Eu3+, and centrifuged at 1000 rpm for 1 minute.2) Incubation at 25° C. for 60 minutes.8) Acquisition of fluorescence signals using Envision 2104 Reader. |
| Affinity data for this assay | |
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