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Related Concept Videos

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: Feb 22, 2026

Single-Molecule Tracking Microscopy - A Tool for Determining the Diffusive States of Cytosolic Molecules
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Two-Color Single-Molecule Tracking in Live Cells.

Siegfried Hänselmann1, Dirk-Peter Herten2

  • 1Physikalisch-Chemisches Institut & CellNetworks Cluster, Heidelberg University, Im Neuenheimer Feld 229, 69120, Heidelberg, Germany.

Methods in Molecular Biology (Clifton, N.J.)
|September 20, 2017
PubMed
Summary
This summary is machine-generated.

This study details two-color single-molecule tracking to measure protein-protein interactions in live cell plasma membranes. The method uses SNAP-tag and HaloTag fusion constructs with TIRF microscopy for precise kinetic analysis.

Keywords:
DiffusionDimerizationFluorescence microscopyHaloTagLabeling protocolMembraneProtein-protein interactionsSNAP-tagSignalingSingle-particle tracking

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Area of Science:

  • Cell Biology
  • Biophysics
  • Microscopy

Background:

  • Understanding dynamic cellular processes like signaling requires measuring protein-protein interactions in live cells.
  • Single-molecule tracking offers high sensitivity to cellular heterogeneities and rare events.

Purpose of the Study:

  • To describe the methodology for obtaining two-color single-molecule tracking data of plasma membrane proteins.
  • To outline the data analysis steps for studying protein diffusion and dimerization kinetics.

Main Methods:

  • Utilizing SNAP-tag and HaloTag fusion constructs for protein labeling.
  • Employing total internal reflection fluorescence (TIRF) microscopy for live-cell imaging.
  • Implementing two-color single-molecule tracking for real-time observation of molecular movement.

Main Results:

  • Achieving localization precision beyond the diffraction limit of light.
  • Quantifying diffusion behavior and dimerization kinetics of individual proteins.
  • Enabling the study of protein-protein interactions at a single-event level.

Conclusions:

  • Two-color single-molecule tracking is a powerful technique for dissecting molecular mechanisms in live cells.
  • This method provides detailed insights into protein dynamics and interactions within the plasma membrane.
  • The described protocol facilitates the investigation of complex cellular signaling pathways.