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Assay Method Information |
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| | In Vitro Enzyme Inhibition Assay |
| Description: | Assay Procedures:The abbreviations used in the following assay have the following meanings:HEPES: hydroxyethyl piperazine ethanesulfonic acid;Brij-35: polyoxyethylene lauryl ether;DTT: dithiothreitol;Coating Reagent #3: #3 coating agent;EDTA: ethylene diamine tetraacetic acid, purchased from Sigma Co. Ltd.;FAM labeled peptide: fluorescein labeled peptide 22 (GL Biochem);ATP: adenosine triphosphate (Sigma);DMSO: dimethyl sulfoxide;EGFR: human epidermal growth factor receptor (Carna);HER2: human epidermal growth factor receptor 2 (Carna);HER4: human epidermal growth factor receptor 4 (Carna).1. Formulating the agents to be used in the assay(1) 1.25-fold MnCl2-free kinase buffer (62.5 mM HEPES, PH 7.5, 0.001875% Brij-35, 12.5 mM MgCl2, 2.5 mM DTT);(2) 1.25-fold MnCl2-containing kinase buffer (62.5 mM HEPES, pH 7.5, 0.001875% Brij-35, 12.5 mM MgCl2, 12.5 mM MnCl2, 2.5 mM DTT);(3) Stop buffer (100 mM HEPES, pH 7.5, 0.015% Brij-35, 0.2% Coating Reagent #3, 50 mM EDTA);(4) 2.5-fold kinase solutions (to the 1.25-fold kinase buffers were added the corresponding kinases to formulate 2.5-fold EGFR, HER2, HER4 kinase solutions);(5) 2.5-fold peptide solutions (to the 1.25-fold kinase buffers were added FAM labeled peptide and ATP to formulate the peptide solutions);(6) 5-fold compound solutions (using 100% DMSO to formulate 50-fold compound solutions having different concentration gradients, and diluting with water by 10 times to obtain 5-fold compound solutions having different concentration gradients);2. Adding 5 μL of a 5-fold compound solution to a 384-well plate;3. Adding 10 μL of a 2.5-fold kinase solution to incubate for 10 min;4. Then adding 10 μL of a 2.5-fold peptide solution, and reacting at 28° C. for 1 h; and5. Finally, adding 25 μL of stop buffer to terminate the reaction, and reading the data with Caliper.6. Curve fitting to obtain an IC50 value. |
| Affinity data for this assay | |
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