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Updated: Jun 11, 2026
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Dual-Color Fluorescence Cross-Correlation Spectroscopy to Study Protein-Protein Interaction and Protein Dynamics in Live Cells
Published on: December 11, 2021
Fluorescence lifetime cross correlation spectroscopy resolves EGFR and antagonist interaction in live cells
1Birck Nanotechnology & Bindley Bioscience Center, Department of Agricultural & Biological Engineering, Purdue University, West Lafayette, Indiana 47906, USA.
Analytical Chemistry
|July 1, 2010
Summary
Fluorescence Lifetime Correlation Spectroscopy (FLCS) now differentiates biomolecules by lifetime, overcoming limitations of FCS and FCCS. This technique successfully monitored antibody-receptor interactions in live cells, revealing EGFR internalization.
Area of Science:
- Biophysics
- Molecular Biology
- Cell Biology
Background:
- Fluorescence Correlation Spectroscopy (FCS) and Cross-Correlation Spectroscopy (FCCS) are powerful single-molecule techniques for studying biomolecular interactions and dynamics.
- However, FCS struggles to differentiate species of similar molecular weight, and FCCS faces challenges with volume overlap and spectral crosstalk.
- These limitations restrict their application in complex biological systems.
Purpose of the Study:
- To introduce Fluorescence Lifetime Correlation Spectroscopy (FLCS) as a novel method for analyzing biomolecular interactions in vitro and in vivo.
- To demonstrate FLCS's ability to overcome the limitations of traditional FCS and FCCS.
- To investigate the interaction of an antagonist antibody with the epidermal growth factor receptor (EGFR) in live cells.
Main Methods:
- Utilized FLCS with a single laser source and detector to separate autocorrelation functions based on fluorescence lifetime.
- Performed in vitro experiments to determine the dissociation constant (Kd) of labeled IgG and anti-IgG.
- Applied FLCS, fluorescence lifetime cross-correlation, and fluorescence lifetime imaging (FLIM) to study EGFR-antibody interactions in live HEK293 cells.
Main Results:
- Successfully demonstrated in vitro interaction of labeled IgG and anti-IgG with a Kd of 11 ± 3 nM, separated by lifetime.
- Quantified the in vivo interaction of an EGFR neutralizing antibody with EGFR-GFP in live cells, yielding a Kd of 9.2 ± 2.7 nM.
- Observed a significant decrease in EGFR density (from 540 to 38 receptors/µm²) upon antibody addition, indicating receptor internalization.
Conclusions:
- FLCS is a viable technique for monitoring biomolecular interactions, offering enhanced specificity through lifetime discrimination.
- The study provides quantitative insights into antibody-EGFR interactions and receptor internalization mechanisms in live cells.
- FLCS opens new avenues for studying protein-protein interactions and cellular responses in complex biological environments.
