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

Clocked stepping of an artificial protein walker along a DNA track.

Nature nanotechnology·2026
Same author

Evaluation and telemetry-based detection of GPS spoofing effects on UAV navigation using software-defined radio.

Scientific reports·2026
Same author

Comparative study of cemented versus screwed prosthesis on the marginal bone stability around dental implants.

Bioinformation·2026
Same author

Malpractice Litigation in Spinal Surgery: Lessons From Real-World Cases and Recommendations for Risk Reduction.

Clinical spine surgery·2025
Same author

Drug Development.

Alzheimer's & dementia : the journal of the Alzheimer's Association·2025
Same author

Targeting non-coding RNAs to overcome resistance and improving outcomes in glioblastoma.

Global medical genetics·2025

Related Experiment Video

Updated: Mar 14, 2026

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide
07:33

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Published on: December 19, 2020

7.3K

A Quantitative Fluorescence-Based Assay for Assessing LIM Domain-Peptide Interactions.

Neil O Robertson1, Manan Shah1, Jacqueline M Matthews2

  • 1School of Life and Environmental Sciences, The University of Sydney, NSW, 2006, Australia.

Angewandte Chemie (International Ed. in English)
|September 21, 2016
PubMed
Summary

New Förster resonance energy transfer (FRET) experiments quantify protein interactions. These assays measure binding affinity and rates for transcriptional regulators and their disordered partners, overcoming previous aggregation challenges.

Keywords:
FRETfluorescencekineticsprotein engineeringprotein-protein interactions

More Related Videos

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes
09:12

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Published on: December 13, 2019

8.5K
PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions
10:58

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Published on: July 27, 2017

10.0K

Related Experiment Videos

Last Updated: Mar 14, 2026

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide
07:33

Fluorescent Leakage Assay to Investigate Membrane Destabilization by Cell-Penetrating Peptide

Published on: December 19, 2020

7.3K
A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes
09:12

A Quantitative Fluorescence Microscopy-based Single Liposome Assay for Detecting the Compositional Inhomogeneity Between Individual Liposomes

Published on: December 13, 2019

8.5K
PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions
10:58

PIP-on-a-chip: A Label-free Study of Protein-phosphoinositide Interactions

Published on: July 27, 2017

10.0K

Area of Science:

  • Biochemistry
  • Molecular Biology
  • Protein-Protein Interactions

Background:

  • Transcriptional regulators are obligate binding proteins that aggregate without partners, complicating interaction studies.
  • Evaluating binding affinity and kinetics for such proteins has been challenging.

Purpose of the Study:

  • To develop and validate Förster resonance energy transfer (FRET)-based assays for measuring binding affinity, off-rates, and inferred on-rates.
  • To characterize interactions between a family of transcriptional regulators and their intrinsically disordered binding partners.

Main Methods:

  • Utilized fusion constructs with binding domains linked by a protease-cleavable tether and fluorescent proteins.
  • Monitored Förster resonance energy transfer (FRET) signal loss upon tether cleavage, followed by dilution or peptide competition.
  • Quantified binding affinities ranging from 10-9 to 10-5 M.

Main Results:

  • Successfully measured binding affinities and kinetic parameters for multiple protein interactions.
  • Demonstrated the utility of FRET-based assays for studying aggregation-prone proteins.
  • Revealed significant differences in binding properties between closely related protein interactions.

Conclusions:

  • Developed a robust FRET-based methodology for characterizing protein-protein interactions, particularly for aggregation-prone proteins.
  • The findings highlight the diverse binding properties even among related transcriptional regulator and intrinsically disordered partner interactions.
  • This approach provides a valuable tool for quantitative analysis of biomolecular interactions.