2. Academic Validation
  3. Automated FRET Two-Hybrid Analysis

Automated FRET Two-Hybrid Analysis

  • J Biophotonics. 2025 Apr 23:e70033. doi: 10.1002/jbio.70033.
Zhiqiang Wei 1 2,  Yanling Xu 1 2,  Jingzhen Wang 1 2,  Beini Sun 1 2,  Qialing Huang 1 2,  Zhengfei Zhuang 1 2,  Tongsheng Chen 1 2,  Min Hu 1 2
Affiliations

Affiliations

  • 1 MOE Key Laboratory of Laser Life Science & Institute of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou, Guangdong, China.
  • 2 Guangdong Provincial Key Laboratory of Laser Life Science, College of Biophotonics, School of Optoelectronic Science and Engineering, South China Normal University, Guangzhou, Guangdong, China.
PMID: 40268497 DOI: 10.1002/jbio.70033
Abstract

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 C 32 V = 0.30 ± 0.01 $$ {E}_D^{\mathrm{C}32\mathrm{V}}=0.30\pm 0.01 $$ , S C 32 V = 1.06 ± 0.14 $$ {S}^{\mathrm{C}32\mathrm{V}}=1.06\pm 0.14 $$ ; CVC: E D CVC = 0.40 ± 0.02 $$ {E}_D^{\mathrm{CVC}}=0.40\pm 0.02 $$ , S CVC = 1.90 ± 0.11 $$ {S}^{\mathrm{CVC}}=1.90\pm 0.11 $$ ) matched reference values. Applied to Bcl-xL/Bak interactions under A1331852 treatment, LURS revealed dose-dependent stoichiometry reduction ( 1.87 → 1.12 $$ 1.87\to 1.12 $$ ). The method achieved precise signal extraction while preserving native cellular conditions, overcoming throughput constraints in dynamic protein interaction studies.

Keywords

FRET quantitative analysis; FRET two‐hybrid assay; automated data processing; image processing; stoichiometry.

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