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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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Oligomerization Dynamics of Cell Surface Receptors in Living Cells by Total Internal Reflection Fluorescence Microscopy Combined with Number and Brightness Analysis
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Combined FCS and PCH Analysis to Quantify Protein Dimerization in Living Cells.

Laura M Nederveen-Schippers1, Pragya Pathak1, Ineke Keizer-Gunnink1

  • 1Department of Cell Biochemistry, University of Groningen, 9747 AG Groningen, The Netherlands.

International Journal of Molecular Sciences
|July 24, 2021
PubMed
Summary

This study introduces a new method, brightness and diffusion global analysis (BDGA), to accurately measure protein dimerization in living cells. The technique uses fluorescence fluctuation spectroscopy to quantify dimer presence, overcoming previous detection challenges.

Keywords:
Dictyostelium discoideumFK506 binding protein 12GFPbrightness and diffusion global analysisdimeric proteinfluorescence correlation spectroscopyfluorescence fluctuation spectroscopyphoton counting histogram

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Area of Science:

  • Biophysics
  • Cell Biology
  • Molecular Biology

Background:

  • Protein dimerization is vital for cellular regulation.
  • Detecting protein dimers in living cells remains a significant challenge.

Purpose of the Study:

  • To develop and validate a robust methodology for measuring protein dimerization in real-time within living cells.
  • To adapt fluorescence fluctuation spectroscopy for quantitative analysis of protein-protein interactions.

Main Methods:

  • Utilized a combination of fluorescence correlation spectroscopy (FCS) and photon counting histogram (PCH) analysis.
  • Developed the brightness and diffusion global analysis (BDGA) method for single-color fluorescence fluctuation data.
  • Validated the method using cell lysates with known GFP and tandem-dimer GFP (diGFP) ratios and applied it to ligand-induced dimerization of FKBP12-GFP.

Main Results:

  • Demonstrated that average particle brightness directly correlates with the fraction of dimer present.
  • Successfully distinguished between monomeric GFP, dimeric diGFP, and ligand-induced dimerization of FKBP12-GFP in living cells.
  • Showcased the robustness and accuracy of the BDGA method for studying cytosolic protein dimerization.

Conclusions:

  • The brightness and diffusion global analysis (BDGA) offers a reliable approach for investigating protein dimerization in living cells.
  • This method provides a significant advancement over existing techniques, which are often prone to errors.
  • The developed methodology is broadly applicable to the study of any cytosolic protein dimerization using single-color fluorescence fluctuation spectroscopy.