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Updated: Jun 2, 2026
Detecting the Ligand-binding Domain Dimerization Activity of Estrogen Receptor Alpha Using the Mammalian Two-Hybrid Assay
Published on: December 19, 2018
Single-label time-resolved luminescence assay for estrogen receptor--ligand binding
Roope Huttunen1, Shweta, Eija Martikkala
1Laboratory of Biophysics, Department of Cell Biology and Anatomy and Medicity Research Laboratories, Institute of Biomedicine, University of Turku, 20520 Turku, Finland. roohuttu@utu.fi
A novel single-label assay using quenching resonance energy transfer (QRET) offers a simpler, more sensitive method for screening nuclear receptor regulators. This method enhances high-throughput screening by improving signal-to-background ratios compared to traditional assays.
Area of Science:
- Biochemistry
- Molecular Biology
- Assay Development
Background:
- High-throughput screening (HTS) requires sensitive, homogeneous luminescence-based microplate assays for nuclear receptor regulators.
- Time-resolved fluorescence resonance energy transfer (TR-FRET) offers high sensitivity but involves complex, expensive dual-labeling methods.
Purpose of the Study:
- To develop a simplified, single-label assay for estrogen receptor (ER) ligand binding.
- To overcome the limitations of multi-label TR-FRET assays for nuclear receptor screening.
Main Methods:
- Developed a quenching resonance energy transfer (QRET) assay using a single-labeled estradiol (Eu-E2) and soluble quenchers.
- Measured luminescence signal changes upon ER-ligand binding, where binding protects Eu-E2 from quenching.
- Compared QRET assay performance against a commercial fluorescence polarization (FP) assay.
Main Results:
- QRET assay demonstrated significantly higher signal-to-background (S/B) ratios (5.8-9.2) compared to FP assay (1.3-1.5) for various ER ligands.
- Calculated a K(d) of 30nM for Eu-E2 binding to ER, indicating high affinity.
- The assay successfully differentiated between binders and non-binders, showing displacement with increasing competitor concentrations.
Conclusions:
- QRET represents a novel, efficient single-label assay format for nuclear receptor ligand screening.
- This method provides a cost-effective and less complex alternative to multi-label assays like TR-FRET.
- The enhanced sensitivity and signal-to-background ratio make QRET suitable for HTS applications.
