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Assay Method Information

Assay Name:  IC50 measurements for inhibitors using BRD9 AlphaLisa Binding Assay
Description:  His/Flag epitope tagged BRD9134-239 was cloned, expressed, and purified to homogeneity. BRD9 binding and inhibition was assessed by monitoring the engagement of biotinylated H4-tetraacetyl peptide (New England Peptide, NEP2069-11/13) with the target using the AlphaLisa technology (Perkin-Elmer). Specifically, in a 384 well ProxiPlate BRD9 (50 nM final) was combined with peptide (3 nM final) in 50 mM HEPES (pH 7.5), 150 mM NaCl, 1 mM TCEP, 0.01% (w/v) BSA, and 0.008% (w/v) Brij-35 either in the presence of DMSO (final 0.8% DMSO) or compound dilution series in DMSO. After 20 minutes incubation at room temperature AlphaLisa Streptavidin Acceptor Beads (Perkin-AL125C) and AlphaLisa Nickel donor beads (Perkin AS 10 ID) were added to a final concentration of 15 ug/mL each. After ninety minutes of equilibration in the dark, the plates were read on an Envision instrument and IC50s calculated using a four parameter non-linear curve fit.
Affinity data for this assay
 

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Last update November 1, 2007
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