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| | AlphaScreen BRD Binding Assay |
| Description: | Assays were performed with minor modifications from the manufacturer's protocol (PerkinElmer, USA). All reagents were diluted in 50 mM HEPES, 150 mM NaCl, 0.1% w/v BSA, 0.01% w/v Tween20, pH 7.5, and allowed to equilibrate to room temperature prior to addition to plates. After addition of Alpha beads to master solutions, all subsequent steps were performed in low light conditions. A 2× solution of components with final concentrations of BRD at 40 nM, Ni-coated acceptor bead at 25 ug/mL, and 20 nM biotinylated-JQ1 was added in 10 uL to 384-well plates (AlphaPlate-384, PerkinElmer, USA). After a 1 min 1000 rpm spin-down, 100 nL of compounds in DMSO from stock plates were arrayed in duplicate dose response by pin transfer using a Janus Workstation (PerkinElmer, USA). The streptavidin-coated donor beads (25 ug/mL final) were added as with previous solution in a 2×, 10 uL volume. Following this addition, the plates were sealed with foil to block light exposure and to prevent evaporatio |
| Affinity data for this assay | |
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