| Assay Method Information | |
| | In Vitro Activity Assay |
| Description: | Table 1: 1. In Vitro Evaluation of Inhibitory Activity of Compounds Disclosed Herein Against MNK2 Protein KinasePurpose of experiment: to test the inhibitory activity of the compounds against MNK2 protein kinaseExperimental materials: assay buffer: 8 mM 3-(N-morpholino)propanesulfonic acid, 0.2 mM disodium ethylenediaminetetraacetate, 0.01% polyoxyethylene lauryl ether, 5% glycerol, 0.1% β-mercaptoethanol and 1 mg of bovine serum albuminExperimental operation: Mnk2 protein kinase inhibitory activity assays were performed using the KinaseProfiler™ service from Eurofins Pharma Discovery Services UK Limited. Serially diluted DMSO solutions containing the compounds to be tested (3-fold serial dilution, starting from 10 μM), MNK2 (h) protein kinase and 0.33 mg/mL myelin basic protein were added to a freshly prepared buffer (pH 7.0), and then stirred homogeneously. The reaction was initiated by adding a mixture of 33P-ATP (intensity of radioactivity: 10 μCi/μL) and 10 mM magnesium acetate. After the resulting mixture was reacted at room temperature for 40 min, the reaction was terminated by adding phosphoric acid to dilute to a concentration of 0.5%. 10 μL of the reaction solution was filtered using a P30 filtermat, and then the filtermat was washed four times with 0.425% phosphoric acid for 4 min each, followed by washing once with methanol. After drying, the intensity of radioactivity was determined using the Filter-Binding method. |
| Affinity data for this assay | |
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