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A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Published on: December 1, 2018

High-throughput fluorescence assay for membrane-protein interaction.

Hyunjin Kim¹, Hamid Samareh Afsari, Wonhwa Cho

¹Department of Chemistry, University of Illinois at Chicago, Chicago, IL 60607.

Journal of Lipid Research

|September 6, 2013

Summary

Researchers developed a new fluorescence assay to measure how proteins bind to membranes. This high-throughput method uses enhanced green fluorescence protein (EGFP) and a special lipid, enabling rapid and sensitive analysis of protein-lipid interactions.

Keywords:

dark quencher green fluorescence protein high-throughput assay membrane-protein binding

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Last Updated: May 8, 2026

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

Published on: December 1, 2018

Protein Membrane Overlay Assay: A Protocol to Test Interaction Between Soluble and Insoluble Proteins in vitro

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Published on: August 14, 2011

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Measuring Transcellular Interactions through Protein Aggregation in a Heterologous Cell System

Published on: May 22, 2020

Area of Science:

Biochemistry
Molecular Biology
Biophysics

Background:

Membrane-protein interactions are crucial for cellular functions.
Existing methods for measuring protein-membrane binding lack universality and high-throughput capability.

Purpose of the Study:

To develop a robust, high-throughput fluorescence assay for quantifying protein-membrane interactions.
To enable sensitive and accurate determination of lipid specificity and binding affinity.

Main Methods:

Utilized enhanced green fluorescence protein (EGFP)-fusion proteins.
Employed vesicles containing N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE), a lipid quencher.
Measured fluorescence quenching of EGFP upon binding to dabsyl-PE-containing vesicles.

Main Results:

Demonstrated a significant decrease (>50%) in EGFP fluorescence intensity upon protein-vesicle binding.
Successfully determined lipid specificity and affinity for multiple lipid-binding domains (PH, ENTH, PX).
Validated the assay's applicability for high-throughput screening of modulators of membrane binding.

Conclusions:

The developed fluorescence quenching assay is a simple, rapid, sensitive, and accurate tool for studying protein-membrane interactions.
This assay is universally applicable and suitable for high-throughput screening.
Facilitates the study of various lipid-binding domains and the discovery of small molecule modulators.