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Protein Dynamics in Living Cells

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Related Experiment Video

Updated: Nov 6, 2025

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

Published on: April 22, 2021

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission.

György Vámosi¹, Sarah Miller², Molika Sinha²

¹Department of Biophysics and Cell Biology, Faculty of Medicine, University of Debrecen.

Journal of Visualized Experiments : Jove

|May 10, 2021

Summary

This study presents a new protocol and algorithm for quantifying Förster Resonance Energy Transfer (FRET) efficiency in living cells. It accurately measures energy transfer by assessing donor quenching and acceptor emission, crucial for studying protein interactions.

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Last Updated: Nov 6, 2025

Assessing Protein Interactions in Live-Cells with FRET-Sensitized Emission

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Published on: April 22, 2021

Imaging Protein-protein Interactions in vivo

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Published on: October 10, 2010

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Published on: May 26, 2014

Area of Science:

Biophysics
Cell Biology
Microscopy

Background:

Förster Resonance Energy Transfer (FRET) is a biophysical technique to study molecular interactions.
FRET efficiency depends on distance, orientation, and spectral overlap between donor and acceptor molecules.
Existing FRET measurement algorithms often lack comprehensive protocols for critical parameter assessment.

Purpose of the Study:

To provide a detailed protocol and algorithm for quantifying FRET efficiency in living cells.
To enable accurate measurement of sensitized acceptor emission and donor quenching.
To address the need for precise FRET quantification in live-cell imaging.

Main Methods:

Development of a novel algorithm for ratiometric FRET quantification.
Detailed protocol for assessing spectral crosstalk (bleed-through) of fluorescent proteins.
Method for determining the detection efficiency of the microscopy setup.

Main Results:

A robust method for quantifying FRET efficiency based on donor quenching and acceptor emission.
Accurate assessment of critical parameters like spectral crosstalk and detection efficiency.
Validation of the protocol for live-cell FRET studies.

Conclusions:

The provided protocol and algorithm enable precise FRET efficiency quantification in living cells.
Accurate assessment of crosstalk and detection efficiency is vital for reliable FRET measurements.
This method facilitates advanced studies of protein-protein interactions within subcellular compartments.