Jove
Visualize
Contact Us
JoVE
x logofacebook logolinkedin logoyoutube logo
ABOUT JoVE
OverviewLeadershipBlogJoVE Help Center
AUTHORS
Publishing ProcessEditorial BoardScope & PoliciesPeer ReviewFAQSubmit
LIBRARIANS
TestimonialsSubscriptionsAccessResourcesLibrary Advisory BoardFAQ
RESEARCH
JoVE JournalMethods CollectionsJoVE Encyclopedia of ExperimentsArchive
EDUCATION
JoVE CoreJoVE BusinessJoVE Science EducationJoVE Lab ManualFaculty Resource CenterFaculty Site
Terms & Conditions of Use
Privacy Policy
Policies

Related Concept Videos

Protein Dynamics in Living Cells01:19

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...

You might also read

Related Articles

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

Sort by
Same author

Stringent selection drives convergence toward omicron-like SARS-CoV-2 receptor-binding motifs.

Nature communications·2026
Same author

Electrostatic interactions constrain generalization of porous-media models for intracellular diffusion in mammalian cells.

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

IRF1 is a context-dependent homeostatic gatekeeper of basal immunity and antiviral readiness.

The Journal of biological chemistry·2026
Same author

The natural herbicide rhein targets photosystem I.

Scientific reports·2024
Same author

Reversing protonation of weakly basic drugs greatly enhances intracellular diffusion and decreases lysosomal sequestration.

eLife·2024
Same author

Promiscuous Janus kinase binding to cytokine receptors modulates signaling efficiencies and contributes to cytokine pleiotropy.

Science signaling·2024

Related Experiment Video

Updated: May 25, 2026

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates
06:48

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Published on: January 5, 2024

Protein-binding dynamics imaged in a living cell.

Yael Phillip1, Vladimir Kiss, Gideon Schreiber

  • 1Department of Biological Chemistry, Weizmann Institute of Science, Rehovot 76100, Israel.

Proceedings of the National Academy of Sciences of the United States of America
|February 7, 2012
PubMed
Summary

In vivo protein binding rates closely match in vitro measurements, even within crowded cells. This study validates using in vitro data to understand cellular protein interactions.

More Related Videos

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking
16:21

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Published on: March 10, 2014

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques
08:28

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Published on: November 2, 2018

Related Experiment Videos

Last Updated: May 25, 2026

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates
06:48

Single-Molecule Measurement of Protein Interaction Dynamics Within Biomolecular Condensates

Published on: January 5, 2024

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking
16:21

Visualizing Protein-DNA Interactions in Live Bacterial Cells Using Photoactivated Single-molecule Tracking

Published on: March 10, 2014

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques
08:28

Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques

Published on: November 2, 2018

Area of Science:

  • Biochemistry
  • Cell Biology
  • Biophysics

Background:

  • In vitro determined rate constants for protein interactions are often extrapolated to in vivo biological systems.
  • The validity of in vitro data for predicting in vivo protein binding dynamics remains uncertain, especially considering cellular complexity.

Purpose of the Study:

  • To measure protein-protein binding kinetics in living cells and compare them to in vitro data.
  • To assess the impact of the intracellular environment, including macromolecular crowding, on binding rate constants.
  • To determine if in vitro binding data can be reliably extrapolated to intracellular processes.

Main Methods:

  • Measuring protein-protein binding dynamics in living HeLa cells using microinjection and Förster resonance energy transfer (FRET).
  • Quantifying association rate constants for wild-type and mutant proteins.
  • Utilizing fluorescence recovery after photobleaching (FRAP) for single-cell analyses of binding kinetics and cell density.

Main Results:

  • In vivo association rate constants were only slightly lower (25-50%) than in vitro values for wild-type and optimized mutants.
  • A slower-binding mutant showed no change in its rate constant in vivo compared to in vitro.
  • Cellular density variations significantly impacted binding rates, causing up to a twofold effect.
  • The intracellular environment exerted a minimal influence on the association kinetics of this model protein interaction.

Conclusions:

  • The intracellular environment has a limited effect on protein-protein association kinetics.
  • In vitro determined binding rate constants are largely applicable to in vivo biological processes.
  • This study provides justification for extrapolating in vitro binding data to understand cellular mechanisms.