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Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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Different fluorescence-based techniques are used to study the protein dynamics in living cells. These techniques include FRAP, FRET, and PET.
Fluorescent recovery after photobleaching (FRAP) is a fluorescent-protein-based detection technique used to quantify protein movement rates within the cell. This method exposes a small portion of the cell to an intense laser beam. The laser beam causes permanent photobleaching of the fluorophore-tagged proteins in the exposed region. As the bleached...
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Related Experiment Video

Updated: May 9, 2025

Automated Two-dimensional Spatiotemporal Analysis of Mobile Single-molecule FRET Probes
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Measuring Molecular Interactions with Subcellular Resolution: Single-Cell FRET Using the Quantitative Three-Filterset

István Rebenku1, Cameron B Lloyd1, György Vereb1,2,3

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

Methods in Molecular Biology (Clifton, N.J.)
|April 30, 2025
PubMed
Summary
This summary is machine-generated.

This study details a method for measuring molecular interactions using quantitative Förster resonance energy transfer (FRET). The protocol enables accurate FRET efficiency mapping and analysis of molecular expression levels in cellular processes.

Keywords:
EGFRFRET microscopyFluorescence resonance energy transferFörster resonance energy transfer (FRET)Integrin A5Molecular interactionsMolecular proximitySingle-cell pixel-by-pixel FRET analysis

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Area of Science:

  • Cellular Biology
  • Biophysics
  • Biochemistry

Background:

  • Molecular interactions, not just expression levels, are crucial for cellular signaling.
  • Förster resonance energy transfer (FRET) is a sensitive technique for measuring molecular proximity (2-10 nm).
  • Quantitative FRET, specifically spectral spillover-corrected methods, offers robust measurements of molecular interactions.

Purpose of the Study:

  • To provide a comprehensive protocol for implementing quantitative FRET measurements.
  • To enable accurate assessment of molecular interactions and expression levels in cellular processes.
  • To introduce the RiFRET ImageJ/Fiji plugin for streamlined FRET data analysis.

Main Methods:

  • Utilizing spectral spillover-corrected quantitative (three-filterset) FRET.
  • Applying the method in flow cytometry and fluorescence microscopy.
  • Employing the freely available RiFRET plugin for image processing and FRET map derivation.

Main Results:

  • Pixel-by-pixel quantitative FRET efficiency values are obtained.
  • FRET-corrected fluorescent intensities reflecting molecular expression levels are derived.
  • The RiFRET plugin facilitates user-friendly and automated processing of large image datasets.

Conclusions:

  • Quantitative FRET provides valuable insights into molecular interactions beyond simple expression levels.
  • The presented protocol and RiFRET plugin offer a versatile and accessible approach for FRET analysis.
  • This method enhances the understanding of cellular signaling pathways through precise molecular interaction studies.