Figure 1. Schematic depiction of a competitive AlphaScreen assay.

When His₆-tagged recombinant protein (BRD4(1)) binds to biotinylated probe (Bio-JQ1), streptavidin-coated donor beads and nickel-coated acceptor beads are brought into close proximity (<200 nm). Upon illumination at 680 nm, the photosensitized donor bead transfers singlet state oxygen (¹Δg O₂) to the acceptor bead, initializing a series of reactions resulting in a fluorescent signal at 520-620 nm. Incubation with an inhibitor can disrupt the protein-probe complex, resulting in loss of signal.