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Assay Method Information |
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| | Competition Binding Assay |
| Description: | For the binding assays, streptavidin-coated magnetic beads were treated with biotinylated affinity ligands for 30 min at room temperature to generate affinity resins. The liganded beads were blocked with excess biotin and washed with blocking buffer (SeaBlock (Pierce), 1% BSA, 0.05% Tween 20, 1 mM DTT) to remove unbound ligand and to reduce non-specific binding. Binding reactions were assembled by combining kinase, liganded affinity beads, and test compounds in 1x binding buffer (20% SeaBlock, 0.17xPBS, 0.05% Tween 20, 6 mM DTT). Test compounds were prepared as 100x stocks in DMSO and rapidly diluted into the aqueous environment. DMSO was added to control assays lacking a test compound. Primary screen interactions were performed in polypropylene 384-well plates in a final volume of 34 uL, while Kd determinations were performed in polystyrene 96-well plates in a final volume of 135 uL. The assay plates were incubated at room temperature with shaking for 1 hr, long enough for binding reactions to reach equilibrium, and the affinity beads were washed extensively with wash buffer (1xPBS, 0.05% Tween 20) to remove unbound protein. The beads were then resuspended in elution buffer (1xPBS, 0.05% Tween 20, 2 uM non-biotinylated affinity ligand) and incubated at room temperature with shaking for 30 min. The kinase concentration in the eluates was measured by quantitative PCR. Each kinase was tested individually against each compound. |
| Affinity data for this assay | |
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