| Assay Method Information | |
| | ADP-Glo Kinase Assay |
| Description: | To measure the effect of the compounds on the activity of AURKA, the IC50 of the compounds was determined with the ADP-Glo Kinase Assay (Promega, V9101) and the Aurora A Kinase Enzyme System (Promega, V1931). The assay was performed according to the manufacturer's instructions in a white 384-well plate format (Corning Assay Plate, LOT 32216024) by using 1.4 ng/μl Aurora A, 25 mM ATP and 0.1 μg/μl myelin basic protein (MBP) substrate in a total volume of 6 μl (transferred with Integra viaflo 96/384 handheld channel pipette). To generate a dose-response curve, a 3-fold serial dilution of the compounds was tested starting from 1 μM. The kinase was pre-incubated for 10 min at RT with the respective compound, before the substrate mix containing MBP and ATP was added. After 60 min at RT, 6 μl of the ADP-Glo reagent were added and incubated for another 60 min at RT. Afterwards 12 μl of the detection reagent were added and incubated for 30 min at RT before the luminescence was measured with an integration time of 500 ms by using a Filter Max F5 multi-mode microplate reader from Molecular Devices. As a negative control, the reaction was performed with MBP, ATP and Aurora A only. The positive control was performed with inactive Aurora A (boiled for 10 min), MBP and ATP. |
| Affinity data for this assay | |
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