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Protein Dynamics in Living Cells

Protein Dynamics in Living Cells

Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...

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Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

Time-Resolved Fluorescence Anisotropy from Single Molecules for Characterizing Local Flexibility in Biomolecules

Published on: April 25, 2025

Fluorescence anisotropy: from single molecules to live cells.

Claudiu C Gradinaru¹, Denys O Marushchak, Masood Samim

¹Department of Physics, Institute for Optical Sciences, University of Toronto, Toronto, Canada. claudiu.gradinaru@utoronto.ca

|February 23, 2010

Summary

Fluorescence polarization measures molecular details like size and environment. New single-molecule methods and ensemble assays advance protein analysis and drug discovery.

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Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Published on: October 21, 2016

Area of Science:

Biophysical Chemistry
Analytical Biochemistry
Molecular Biophysics

Background:

Fluorescence polarization is a versatile physical quantity linked to molecular properties such as conformation, orientation, size, and nanoscale environment (viscosity, temperature).
Fluorescence anisotropy is a key technique in analytical biochemistry and chemistry for assessing protein/nucleic acid folding and ligand binding affinity via titration.

Purpose of the Study:

This review focuses on novel multi-parameter single-molecule detection techniques and their bioanalytical uses.
It also highlights the application of ensemble polarization assays for studying protein and aptamer dynamics and for high-throughput drug screening.

Main Methods:

Review of advanced single-molecule detection schemes.
Analysis of ensemble polarization assays for binding and conformational studies.
Exploration of high-throughput screening methodologies.

Main Results:

Demonstration of multi-parameter single-molecule detection for detailed molecular analysis.
Validation of ensemble polarization assays for probing biomolecular dynamics and interactions.
Successful application in high-throughput drug discovery.

Conclusions:

Fluorescence polarization offers a powerful, accessible tool for molecular characterization.
Advanced single-molecule and ensemble techniques significantly enhance bioanalytical capabilities.
Polarization assays are crucial for understanding biomolecular mechanisms and accelerating drug development.