| Assay Method Information | |
| | Kinase-Glo luminescent kinase assay |
| Description: | The sample compounds were dissolved in DMSO and diluted it to 500 μM concentration with DMSO and transferred to a dose plate. The compounds were serially diluted with DMSO in 5 fold concentration. Then each concentration was diluted 10-fold with the reaction buffer to get a 10× final concentration. Transfer the compounds with concentrations ranging from 0.003 μM to 50 μM to EGFR assay plate to determine their kinase activity with a dose of 1 μL/well.The positive control compound Gifitinib was dissolved in DMSO as 10 mM stock solution and diluted it to 100 μM concentration with DMSO. The control compound was serially diluted with DMSO in 5 fold concentration. Then each concentration was diluted 10-fold with the reaction buffer to get a 10× final concentration. Transfer the positive control compound with concentrations ranging from 0.00064 μM to 10 μM to EGFR assay plate to determine their kinase activity with a dose of 1 μL/well.For the HPE (hundred percent effect: No kinase and no compound, but containing ATP, substrate and 1% DMSO) and ZPE (zero percent effect: No compound but containing kinase, ATP, substrate and 1% DMSO) wells, dilute 2 μL DMSO 10-fold with reaction buffer to obtain 10% DMSO solution. Then transfer it to the assay plat, 1 μL/well. The positive control wells contain kinase, ATP, substrate and positive control compound in different concentrations. The sample compound wells contain kinase, ATP, substrate and the compounds to be tested in different concentrations.Preparation of the reagents in need: 4×ATP: dilute ATP 4 times with assay buffer to obtain a working solution; 4× substrate: dilute Poly (glucose: tyrosine) 4 times with assay buffer to obtain a working solution; 2.5×EGFR kinase: dilute EGFR kinase 2.5 times with assay buffer to obtain a working solution.Kinase reaction: Add 10× compounds to the assay plate in a 384-well plate layout, 1 μL/well. For the HPE and ZPE wells, equal volume (1 μL/well) of 10% DMSO was added to the 384-well assay plate; Add 2.5×EGFR kinase into the assay plate in 384-well plate layout, 4 μL/well. For HPE and ZPE wells, an equal volume (4 μL/well) of assay buffer was added to the 384-well assay plate; Centrifuge the assay plate with 1000 rpm for 1 min to mix them; Pre-incubate the assay plate for 30 min at 30° C.; Mix equal volume of 4×ATP and 4× substrate to obtain 2×ATP-substrate mixture which serves as a reaction mixture for the determination of EGFR activity; Add 2×ATP-substrate mixture to the assay plate, 5 μL/well; Centrifuge the assay plate at 1000 rpm for 1 min to mix them; Incubate the assay plate for one hour at 30° C.; Add Kinase glo plus was to each well (10 μL/well), and then incubated the assay plate for 20 min at 27° C.; Read luminescence signal with Envision plate reader.Raw data analysis: The raw data was analyzed by Prism 5.0 and the rate of inhibition was calculated by the following formula: Compound inhibition rate=( compound reading−ZPE)/(HPE−ZPE)*100%. |
| Affinity data for this assay | |
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