Search research articles

ABOUT JoVE

Overview Leadership Blog JoVE Help Center

AUTHORS

Publishing Process Editorial Board Scope & Policies Peer Review FAQ Submit

LIBRARIANS

Testimonials Subscriptions Access Resources Library Advisory Board FAQ

RESEARCH

JoVE Journal Methods Collections JoVE Encyclopedia of Experiments Archive

EDUCATION

JoVE Core JoVE Business JoVE Science Education JoVE Lab Manual Faculty Resource Center Faculty Site

Terms & Conditions of Use

Related Concept Videos

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...

The Equilibrium Binding Constant and Binding Strength

The Equilibrium Binding Constant and Binding Strength

The equilibrium binding constant (Kb) quantifies the strength of a protein-ligand interaction. Kb can be calculated as follows when the reaction is at equilibrium:

You might also read

Related Articles

Articles linked to this work by shared authors, journal, and citation graph.

Sort by

Same author

A physics-informed alternative to Richardson-Lucy deconvolution across SNR regimes without iteration cutoffs.

Nature communications·2026

Same author

Mitochondria directly interact with the nuclear pore complex.

Nature·2026

Same author

Stochastic colonization and host-to-host transmission shape gut bacterial variability.

bioRxiv : the preprint server for biology·2026

Same author

Modulation of Single-Molecule Emission at Hexagonal Boron Nitride Surfaces.

Nano letters·2026

Same author

Resolving fluorescently labeled species using highly multiplexed spectral FLIM.

Scientific reports·2026

Same author

Simulation-based inference captures non-Markovian effects as exemplified in protein production kinetics through cell division.

Proceedings of the National Academy of Sciences of the United States of America·2026

Same journal

The Role of Functional Groups in Substituted Benzoic Acids Used as Dopants in Liquid Crystal Mixtures on the Nematic-Isotropic Transitions.

The journal of physical chemistry. B·2026

Same journal

Hyperfine Coupling Quantifies Hole Delocalization in Triarylamine Radical Cations of D-χ-A Molecules.

The journal of physical chemistry. B·2026

Same journal

A Solvatochromic-Chemometric Framework to Resolve Subtle Polarity Microenvironment Differences in Cycloalkanes Driven by Molecular Conformation and Substituent Effects: A Proof-Of-Concept for Advanced Aviation Fuel Design.

The journal of physical chemistry. B·2026

Same journal

Selective Effects of Backbone Cyclization and Disulfide Bonding as Global Covalent Constraints on the Conformational Ensemble of Sunflower Trypsin Inhibitor-1.

The journal of physical chemistry. B·2026

Same journal

Europium Coordination Structure in Peptide Complexes Resolved with Simulation and X-ray Absorption Spectroscopy.

The journal of physical chemistry. B·2026

Same journal

Competitive Coordination and Structural Evolution of Phenylalanine-Mg<sup>2+</sup> Complexes in Microaqueous Environments: Insights from DFT and Molecular Dynamics Simulations.

The journal of physical chemistry. B·2026

See all related articles

Search research articles

Related Experiment Video

Updated: May 16, 2025

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Published on: August 4, 2015

Bayesian Inference of Binding Kinetics from Fluorescence Time Series.

J Shepard Bryan^1,2, Stanimir Asenov Tashev^3,4, Mohamadreza Fazel^1,2

¹Department of Physics, Arizona State University, Tempe, Arizona 85281, United States.

The Journal of Physical Chemistry. B

|May 7, 2025

Summary

This summary is machine-generated.

This study introduces a new method to accurately measure binding and unbinding rates from fluorescence data, even with noise and photobleaching. The approach reliably infers kinetic rates alongside photobleaching, improving binding kinetics analysis.

More Related Videos

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

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Published on: January 30, 2018

Related Experiment Videos

Last Updated: May 16, 2025

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell

Published on: August 4, 2015

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

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Published on: January 30, 2018

Area of Science:

Biophysics
Biochemistry
Molecular Dynamics

Background:

Analyzing binding kinetics using fluorescence time traces is challenging due to measurement noise and photophysics.
Photobleaching, unlike photoblinking, cannot be easily mitigated and limits current methods for determining binding and unbinding rates.

Purpose of the Study:

To develop a novel method for inferring binding and unbinding rates alongside photobleaching rates from fluorescence intensity traces.
To overcome limitations of existing methods caused by noise and photobleaching in kinetic rate analysis.

Main Methods:

A two-stage process utilizing a hidden Markov model (HMM) to analyze individual regions of interest (ROIs).
Inferring fluorescence intensity levels and state trajectories from each trace using the HMM.
Employing the inferred state trajectories from all ROIs to determine kinetic rates.

Main Results:

The proposed method effectively analyzes noisy fluorescence traces.
It accurately accounts for photobleaching events during kinetic analysis.
Provides reliable uncertainties for the inferred binding kinetics.

Conclusions:

The developed method offers a robust approach to studying binding kinetics in the presence of noise and photobleaching.
Demonstrated effectiveness through simulations and DNA origami binding experiments.
Enhances the accuracy and reliability of fluorescence-based kinetic measurements.