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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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Automated System for Single Molecule Fluorescence Measurements of Surface-immobilized Biomolecules
10:57

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Published on: November 2, 2009

Robust single-molecule approach for counting autofluorescent proteins.

Laurent Cognet1, Catherine Tardin, Marie-Laure Martin Négrier

  • 1Universite Bordeaux, Centre de Physique Moleculaire Optique et Hertzienne, Centre National de la Recherche Scientifique, 351 Cours de la Liberation, 33405 Talence, France. lcognet@u-bordeaux1.fr

Journal of Biomedical Optics
|July 8, 2008
PubMed
Summary

We developed a single-molecule microscopy technique to count autofluorescent proteins in aggregates. This method measures total intensity until photobleaching, overcoming blinking issues and precisely quantifying protein numbers.

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Area of Science:

  • Biophysics
  • Molecular Biology
  • Microscopy

Background:

  • Quantifying proteins in aggregates is challenging, especially when individual molecules are not optically resolvable.
  • Autofluorescent proteins, like green fluorescent proteins, exhibit blinking, complicating direct counting.
  • Understanding protein aggregate composition is crucial in various biological processes.

Purpose of the Study:

  • To develop and validate a novel method for quantifying single autofluorescent proteins within aggregates.
  • To overcome the limitations of optical resolution and protein blinking in aggregate analysis.
  • To apply the method for precise characterization of protein aggregate populations.

Main Methods:

  • Utilizing single-molecule microscopy to observe autofluorescent protein aggregates.
  • Measuring the total fluorescence intensity emitted by each aggregate until photobleaching.
  • Analyzing intensity decay curves to determine the number of proteins per aggregate.

Main Results:

  • The method accurately quantifies protein numbers in aggregates, even when individual proteins are not resolved.
  • It successfully overcomes the issue of autofluorescent protein blinking.
  • Precise mean composition (±1 protein) was determined for small aggregates.
  • Average protein counts and population inhomogeneity were assessed for larger aggregates.

Conclusions:

  • This photobleaching-based intensity summation method provides a robust approach for quantifying proteins in aggregates.
  • The technique offers high precision for small aggregates and valuable insights into the heterogeneity of larger ones.
  • It was successfully applied to quantify purified citrine protein multimers, demonstrating its practical utility.