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Related Concept Videos

Protein Networks02:26

Protein Networks

An organism can have thousands of different proteins, and these proteins must cooperate to ensure the health of an organism. Proteins bind to other proteins and form complexes to carry out their functions. Many proteins interact with multiple other proteins creating a complex network of protein interactions.
These interactions can be represented through maps depicting protein-protein interaction networks, represented as nodes and edges. Nodes are circles that are representative of a protein,...

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A High-content Imaging Workflow to Study Grb2 Signaling Complexes by Expression Cloning
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Published on: October 30, 2012

A multi-functional imaging approach to high-content protein interaction screening.

Daniel R Matthews1, Gilbert O Fruhwirth, Gregory Weitsman

  • 1Division of Cancer Studies, Randall Division of Cell and Molecular Biophysics, King's College London, London, United Kingdom.

Plos One
|April 17, 2012
PubMed
Summary
This summary is machine-generated.

This study introduces a novel high-content screening platform combining fluorescence anisotropy and lifetime imaging for objective, quantitative protein-protein interaction analysis. The validated method enables rapid, wide-field screening of molecular interactions at the nanoscale.

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Last Updated: May 23, 2026

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10:52

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Published on: October 30, 2012

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Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions
08:07

Probing High-density Functional Protein Microarrays to Detect Protein-protein Interactions

Published on: August 2, 2015

Area of Science:

  • Biophysics
  • Cell Biology
  • Biochemistry

Background:

  • Traditional high-content screening relies on subjective phenotypic measures.
  • Functional imaging offers objective quantification for biological assays.
  • There is a need for advanced screening platforms for protein-protein interactions.

Purpose of the Study:

  • To develop and validate a prototype high-content screening platform integrating fluorescence anisotropy and fluorescence lifetime imaging (FLIM).
  • To enable objective, quantitative screening of small molecule libraries for protein-protein interaction assays.
  • To demonstrate the platform's capability in assessing nanoscale protein interactions.

Main Methods:

  • Combined steady-state fluorescence anisotropy imaging with fluorescence lifetime imaging (FLIM).
  • Utilized wide-field, acceptor fluorescence anisotropy imaging to extract Förster Resonance Energy Transfer (FRET) information.
  • Validated methodologies using eGFP and mRFP1 constructs and performed inhibitor screens on Cdc42 FRET biosensor and CXCR4 internalization.

Main Results:

  • Demonstrated high correlation between fluorescence anisotropy and FLIM methodologies for FRET assays.
  • Successfully validated the platform using a small-scale inhibitor screen of a Cdc42 FRET biosensor.
  • Showcased the ability to measure hetero-FRET variations and inhibitors of CXCR4 internalization.

Conclusions:

  • The developed platform provides objective, quantitative screening of protein-protein interactions.
  • The integration of fluorescence anisotropy and FLIM offers a powerful tool for nanoscale interaction analysis.
  • This approach enables rapid, wide-field screening with high correlation to established methods.