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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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Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy
06:51

Confocal Microscopy Reveals Cell Surface Receptor Aggregation Through Image Correlation Spectroscopy

Published on: August 2, 2018

Mapping receptor density on live cells by using fluorescence correlation spectroscopy.

Yan Chen1, Alina C Munteanu, Yu-Fen Huang

  • 1Department of Chemistry, Shands Cancer Center, UF Genetics Institute, University of Florida, Gainesville, FL 32611, USA.

Chemistry (Weinheim an Der Bergstrasse, Germany)
|April 11, 2009
PubMed
Summary

This study uses fluorescence correlation spectroscopy (FCS) and aptamers to accurately measure cell surface receptor densities. This method offers a sensitive approach for understanding molecular interactions and developing new cancer therapies.

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Area of Science:

  • Biophysics
  • Cell Biology
  • Molecular Interactions

Background:

  • Understanding cell membrane receptor dynamics is crucial for cellular function and therapeutic development.
  • Mapping receptor distribution and monitoring ligand-receptor interactions on live cells presents significant challenges.

Purpose of the Study:

  • To develop a sensitive method for mapping cell surface receptor densities and monitoring molecular interactions on live cells.
  • To apply fluorescence correlation spectroscopy (FCS) with aptamers for precise receptor quantification.

Main Methods:

  • Utilized fluorescence correlation spectroscopy (FCS) with fluorophore-labeled aptamers for specific cell-surface receptor detection.
  • Employed aptamer sgc8 to quantify the binding affinity to human protein tyrosine kinase-7 (PTK7).
  • Constructed a cellular model to estimate receptor densities and distributions on live cell membranes.

Main Results:

  • FCS detected aptamers with high sensitivity, down to 2 molecules, ideal for studying molecular interactions.
  • Determined aptamer sgc8 has a high binding affinity (K(d)=790+/-150 pM) to PTK7.
  • Quantified PTK7 receptor densities on CCRF-CEM cells (1300+/-190 receptors/µm²) and HeLa cells (550+/-90 receptors/µm²).

Conclusions:

  • FCS-based aptamer approach provides a highly sensitive, direct, and fast method for estimating membrane receptor densities.
  • This technique is valuable for molecular interaction studies and density estimations in subcellular structures.
  • The method shows promise for applications in drug discovery and cancer biomarker research.