| Assay Method Information | |
| | Kinase Assay |
| Description: | Materials used include HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), Brij-35 (dodecyl polyglycol ether), DTT (dithiothreitol), EDTA (ethylenediamine tetraacetic acid), EGFR (human epidermal growth factor receptor), HER2 (human epidermal growth factor receptor 2), EGFR T790M (human epidermal growth factor receptor T790M mutation), Peptide FAM-P22 (fluorescein-labeled peptide 22), ATP (adenosine triphosphate), DMSO (dimethyl sulfoxide), 96-well plate, 384-well plate, staurosporine, Coating Reagent #3 and so on, all of which are commercially available.1. Preparation of Kinase Base Buffer and Stop Buffer1× Kinase buffer without MnCl2 was prepared from 50 mM HEPES, pH 7.5, 0.0015% Brij-35, 10 mM MgCl2, and 2 mM DTT. Stop buffer was prepared from 100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, and 50 mM EDTA.2. Preparation of the Compounds for Testing KinasesPreparation of the compounds was performed according to the following procedures: (1) the compound to be tested was diluted to the concentration of 500 μM with 100% DMSO which is 50× of the final desired highest inhibitor concentration, and 100 μL of the diluted compound solution was transferred to a well in a 96-well plate; (2) the compound was gradiently diluted by transferring 20 μL original solution to 60 μL of 100% DMSO in the next well and so forth for a total of 10 concentrations; (3) DMSO (100 μL, 100%) was added to two empty wells as a no-compound control and a no-enzyme control in the same 96-well plate, and the plate was marked as source plate; (4) intermediate plate was prepared by transferring 10 μL of each compound from source plate to a new 96-well plate as the intermediate plate, and to each well of the intermediate plate was added 90 μL of 1× Kinase base buffer, then the intermediate plate was mixed for 10 min on shaker; and (5) assay plate was prepared by transferring 5 μL of each well from the 96-well intermediate plate to a 384-well plate in duplicates.3. Kinase ReactionKinase reaction was performed according to the following procedures: (1) 2.5× kinase solution was prepared by adding kinase into 1× kinase base buffer; (2) 2.5× peptide solution was prepared by adding FAM-labeled peptide and ATP into 1× kinase base buffer; (3) 2.5× kinase solution (10 μL) was added to each well of the 384-well assay plate containing 5 μL of compound in 10% DMSO and then the assay plate was incubated at room temperature for 10 minutes; (4) 2.5× peptide solution (10 μL) was added to each well of the 384-well assay plate; and (5) stop buffer (25 μL) was added to stop the kinase reaction after incubation at 28° C. for a specified period of time.4. Data MeasurementMultifunctional microplate reader (BMG LABTECH, PHER Astar FS) with a 320 nM excitation wavelength was used to read and collected the data of by absorbing light at 665 nM and 620 nM, which was converted from the rate of 665 signal/620 signal. |
| Affinity data for this assay | |
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