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Assay Method Information |
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| | AlphaScreen Assay |
| Description: | In brief, compound plates (1 μL at 10 mM highest concentration; 3-fold, 10-point dilutions in DMSO) were diluted in 1X assay buffer (20 mM TRIS pH 8.0, 25 mM NaCl, 2 mM DTT and 0.05% Tween-20) to 1 mM using a Multimek robotic pipettor (Nanoscreen), and 1 μL was spotted into the wells of 384-well low-volume Proxiplates (PerkinElmer). To these plates was added 9 μL of protein−peptide mix in a 1X assay buffer by Multidrop (Thermo) to bring the final compound concentration to 100 μM, and this was incubated for 30 min at RT. Next, 2 μL of a 1:1 mixture of streptavidin-conjugate donor and nickel-chelate acceptor beads (45μg/mL in 1X assay buffer) were added, and the plates were allowed to incubate for an additional 30 min in the dark at RT. After incubation, the plates were read on an EnVision multilabel reader equipped with an HTS AlphaScreen laser (PerkinElmer). |
| Affinity data for this assay | |
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