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Related Concept Videos

Protein Dynamics in Living Cells01:19

Protein Dynamics in Living Cells

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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Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions
10:44

Fluorescence Anisotropy as a Tool to Study Protein-protein Interactions

Published on: October 21, 2016

Fluorescence Anisotropy Applied to Measure Aptamer-Protein Interactions.

Maira Rivera1, Maureen McKeague2,3

  • 1Department of Chemistry, Faculty of Science, McGill University, Montreal, QC, Canada.

Methods in Molecular Biology (Clifton, N.J.)
|July 9, 2026
PubMed
Summary

This study presents a fluorescence anisotropy method to measure nucleic acid aptamer-protein binding affinity. The technique accurately quantifies dissociation constants (KD) for aptamer-protein interactions, like the thrombin-binding aptamer (TBA) and thrombin.

Keywords:
Abasic siteAptamersBinding affinityDissociation constantFluorescence anisotropyFluorescence polarizationG-quadruplexMolecular interactionsNucleic acid modificationProtein-nucleic acid

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Area of Science:

  • Biochemistry
  • Molecular Biology
  • Biophysical Chemistry

Background:

  • Quantifying biomolecular binding affinities is crucial for understanding molecular interactions.
  • Nucleic acid aptamers offer specific binding capabilities for protein targets.
  • Fluorescence anisotropy is a sensitive biophysical technique for monitoring molecular interactions.

Purpose of the Study:

  • To describe a fluorescence anisotropy-based method for determining the binding affinity between nucleic acid aptamers and protein targets.
  • To provide a detailed protocol for this method, using the thrombin-binding aptamer (TBA)-thrombin system as an example.
  • To highlight the broad applicability of this method for various protein-nucleic acid interactions.

Main Methods:

  • Fluorescently labeling a nucleic acid aptamer.
  • Titrating the labeled aptamer with increasing concentrations of its target protein.
  • Monitoring changes in fluorescence anisotropy to generate binding isotherms and calculate the dissociation constant (KD).

Main Results:

  • The fluorescence anisotropy method accurately quantifies the binding affinity (KD) between the thrombin-binding aptamer (TBA) and thrombin.
  • The protocol provides detailed steps for reagent preparation, plate setup, data acquisition, and analysis.
  • Emphasis is placed on minimizing experimental variability for reliable results.

Conclusions:

  • Fluorescence anisotropy is a rapid, sensitive, and accurate technique for quantifying aptamer-protein binding affinities.
  • The described method is broadly applicable to diverse nucleic acid-protein interaction studies.
  • This protocol facilitates the characterization of biomolecular binding interactions.