Automated FRET Two-Hybrid Analysis

The fluorescence resonance energy transfer (FRET) two-hybrid assay enables live-cell detection of biomolecular complexes but faces high-throughput screening (HTS) limitations due to laborious image analysis. We developed an automated platform using the Luminance-Uniformity-based Region of Interest Selection (LURS) algorithm, accelerating processing 12-fold (6 h  $\to$ 30 min) for three-channel FRET imaging. Validation with FRET standards (C32V: $E_D^{C32V}=0.30\pm 0.01$ , $S^{C32V}=1.06\pm 0.14$ ; CVC: $E_D^{\mathrm{CVC}}=0.40\pm 0.02$ , $S^{\mathrm{CVC}}=1.90\pm 0.11$ ) matched reference values. Applied to Bcl-xL/Bak interactions under A1331852 treatment, LURS revealed dose-dependent stoichiometry reduction ( $1.87\to 1.12$ ). The method achieved precise signal extraction while preserving native cellular conditions, overcoming throughput constraints in dynamic protein interaction studies.