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Related Experiment Video

Updated: May 23, 2026

Imaging Protein-protein Interactions in vivo
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Imaging Protein-protein Interactions in vivo

Published on: October 10, 2010

Imaging protein interactions by FRET microscopy: cell preparation for FRET analysis.

Peter J Verveer, Oliver Rocks, Ailsa G Harpur

    CSH Protocols
    |April 10, 2012
    PubMed
    Summary

    This protocol details cell preparation for Förster Resonance Energy Transfer (FRET) analysis in live and fixed cells. Optimized reagents minimize quenching of GFP mutants and fluorescent dyes for improved FRET imaging.

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    Area of Science:

    • Biophysics
    • Cell Biology
    • Molecular Imaging

    Background:

    • Förster Resonance Energy Transfer (FRET) is a powerful technique for studying molecular interactions in cells.
    • Accurate FRET measurements depend on minimizing signal quenching from various factors.
    • Live and fixed cell preparations require specific considerations for maintaining cellular integrity and fluorophore properties.

    Purpose of the Study:

    • To provide a detailed protocol for preparing cells for FRET analysis.
    • To optimize reagent usage for enhanced FRET signal fidelity.
    • To ensure applicability for both live and fixed cell imaging.

    Main Methods:

    • Development of a cell preparation protocol for FRET.
    • Optimization of reagents to reduce quenching of Green Fluorescent Protein (GFP) mutants.

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    Measurement of Force-Sensitive Protein Dynamics in Living Cells Using a Combination of Fluorescent Techniques
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    Last Updated: May 23, 2026

    Imaging Protein-protein Interactions in vivo
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    Published on: October 10, 2010

    FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors
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    FRET Microscopy for Real-time Monitoring of Signaling Events in Live Cells Using Unimolecular Biosensors

    Published on: August 20, 2012

    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

  • Optimization of reagents to reduce quenching of fluorescent dyes.

    • Main Results:

    • A robust protocol for preparing cells for FRET analysis was established.
    • Reagent optimization successfully minimized quenching effects on GFP mutants.
    • Reagent optimization successfully minimized quenching effects on fluorescent dyes.

    Conclusions:

    • The described protocol enables reliable FRET analysis in both live and fixed cells.
    • Minimized quenching leads to more accurate FRET measurements.
    • This method supports advanced cellular and molecular interaction studies using FRET.