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

You might also read

Related Articles

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

Sort by
Same author

Exploring bacteria-surface interactions with a fluorescent membrane tension probe.

Proceedings of the National Academy of Sciences of the United States of America·2025
Same author

Virus Propagation Linked to Exceedingly Rare Gene-Expression Errors: A Single-Molecule Microscopy Demonstration.

ACS chemical biology·2025
Same author

Predictive Biomarkers for Immune Checkpoint Inhibitor Therapy in Advanced Melanomas.

Surgical oncology clinics of North America·2025
Same author

Spatial functional mapping of hypoxia inducible factor heterodimerisation and immune checkpoint regulators in clear cell renal cell carcinoma.

BJC reports·2024
Same author

Determination of Interactive States of Immune Checkpoint Regulators in Lung Metastases after Radiofrequency Ablation.

Cancers·2022
Same author

The fusion of quantitative molecular proteomics and immune-oncology: a step towards precision medicine in cancer therapeutics.

FEBS letters·2022

Related Experiment Video

Updated: Mar 2, 2026

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell
14:09

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

Published on: August 4, 2015

13.0K

Quantifying intracellular equilibrium dissociation constants using single-channel time-resolved FRET.

Gloria de Las Heras-Martínez1, Josu Andrieu1, Banafshé Larijani1,2

  • 1Instituto Biofisika (CSIC, UPV/EHU), Barrio de Sarriena s/n, 48940, Leioa, Spain.

Journal of Biophotonics
|May 10, 2017
PubMed
Summary

Accurately quantifying protein interactions inside cells is difficult. This study introduces a novel single-channel FLIM method to precisely measure the dissociation constant (Kd) for protein interactions in situ.

Keywords:
FLIMFRETKdequilibrium dissociation constantintracellularprotein interactions

More Related Videos

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry
08:26

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

Published on: April 6, 2022

3.0K
Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells
06:48

Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Published on: January 5, 2024

5.5K

Related Experiment Videos

Last Updated: Mar 2, 2026

Fluorescence Biomembrane Force Probe: Concurrent Quantitation of Receptor-ligand Kinetics and Binding-induced Intracellular Signaling on a Single Cell
14:09

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

Published on: August 4, 2015

13.0K
Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry
08:26

Analyzing the Interaction of Fluorescent-Labeled Proteins with Artificial Phospholipid Microvesicles using Quantitative Flow Cytometry

Published on: April 6, 2022

3.0K
Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells
06:48

Author Spotlight: Evaluation of Protein-Condensate Dynamics in Live Human Cells

Published on: January 5, 2024

5.5K

Area of Science:

  • Biophysics
  • Cell Biology
  • Molecular Interactions

Background:

  • Quantifying intracellular protein interactions, specifically the dissociation constant (Kd), is challenging due to variable protein concentrations.
  • Fluorescence Resonance Energy Transfer (FRET) methods often require measuring both donor and acceptor fluorescence, complicating accurate Kd determination.
  • Existing techniques face limitations in precisely measuring protein binding affinities within the complex cellular environment.

Purpose of the Study:

  • To develop and validate a novel method for accurate intracellular Kd quantification using only donor fluorescence lifetime imaging microscopy (FLIM).
  • To exploit the eGFP-mCherry FRET pair's spectroscopic properties for robust Kd measurement in situ.
  • To assess the impact of incomplete labeling and define the application range of the method via Monte Carlo simulations.

Main Methods:

  • Utilizing single-channel FLIM to measure donor fluorescence lifetime.
  • Applying the eGFP-mCherry FRET pair for Förster Resonance Energy Transfer measurements.
  • Employing Monte Carlo simulations to evaluate the influence of incomplete labeling on Kd quantification.
  • Validating the method by measuring the intracellular Kd of 3-phosphoinositide-dependent kinase 1 (PDK1) homodimerization.

Main Results:

  • A robust method was established to determine intracellular Kd using only single-channel FLIM, overcoming limitations of previous FRET-based approaches.
  • The method accurately quantified the homodimerization Kd of the oncogenic protein PDK1 across various cell lines and conditions.
  • Simulations defined the method's applicability range concerning different Kd values and labeling efficiencies.
  • In vitro validation confirmed the accuracy of the in situ measured Kd values.

Conclusions:

  • The developed single-channel FLIM method provides a precise and broadly applicable tool for quantifying intracellular protein-protein interactions.
  • This technique simplifies Kd measurement by eliminating the need for acceptor channel imaging.
  • The study reveals a competitive regulatory mechanism for PDK1 homodimerization, demonstrating the method's utility in uncovering biological insights.