| Assay Method Information | |
| | DiscoverX's KINOMEscan Technology Assay |
| Description: | 1. Preparation of magnetic beads: biotin-labeled small molecule ligand and avidin-coated magnetic beads were allowed to interact at room temperature for 30 minutes, and then added with excess biotin, followed by washing with a blocking solution (1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand.2. Binding reaction: in a 384 plate, 0.02 mL system, DNA-labeled protein, small molecule ligand-bound magnetic beads, and different concentrations of compound to be tested were mixed in a reaction solution (0.17×PBS, 0.05% Tween 20, 6 mM DTT) at room temperature with shaking for 1 hr, to which was added an eluant (1×PBS, 0.05% Tween 20) for elution. Magnetic beads were resuspended by an eluant (1×PBS, 0.05% Tween 20, 0.5 μM non-biotin-labeled small molecule ligand), and shaken at room temperature for 30 min before separating out the eluant.3. Determination of Kd value: the content of protein in the above eluate was determined by qPCR. Eleven 3-fold dilution concentrations were determined for the compound to be tested, with 3 DMSO controls for each concentration, and the Kd value was obtained by curve fitting. |
| Affinity data for this assay | |
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