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Ligand-receptor kinetics measured by total internal reflection with fluorescence correlation spectroscopy
Alena M Lieto1, Randall C Cush, Nancy L Thompson
1Department of Physics and Astronomy, University of North Carolina at Chapel Hill, Chapel Hill, North Carolina, USA.
Biophysical Journal
|October 29, 2003
Summary
Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) was used to study molecular interactions at surfaces. This method successfully characterized the kinetics of fluorescent ligands binding to receptors on planar membranes.
Area of Science:
- Biophysics
- Surface Science
- Analytical Chemistry
Background:
- Total internal reflection fluorescence correlation spectroscopy (TIR-FCS) analyzes molecular dynamics at interfaces.
- Characterizing ligand-receptor interactions is crucial for understanding biological processes.
Purpose of the Study:
- To demonstrate TIR-FCS for studying the kinetics of reversible ligand-receptor associations.
- To quantify interaction parameters at substrate-supported planar membranes.
Main Methods:
- Utilized TIR-FCS to analyze fluorescence fluctuations of labeled IgG interacting with FcgammaRII receptors.
- Varied IgG concentration, receptor density, observation area, and incident intensity.
Main Results:
- Successfully measured IgG surface density, solution concentration, dissociation kinetics, and diffusion rates.
- Observed molecular counts scaled predictably with experimental parameters.
- Kinetic parameters were consistent with previous findings and independent of incident intensity.
Conclusions:
- TIR-FCS is a powerful technique for quantifying molecular interactions at planar membrane interfaces.
- The study provides key kinetic insights into IgG-FcgammaRII interactions.