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Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions
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Biochemistry
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JoVE Journal Biochemistry
Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

Using Three-color Single-molecule FRET to Study the Correlation of Protein Interactions

DOI:
10.3791/56896-v

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11:22 min

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January 30, 2018

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, , ,
1Institute of Physical Chemistry,University of Freiburg, 2Department of Bionanoscience, Kavli Institute of Nanoscience Delft,Delft University of Technology

Chapters

  • 00:05Title
  • 00:37Total Internal Reflection Fluorescence Microscope (TIRFM) Flow Chamber Assembly
  • 02:15Three-color smFRET Measurement of Hsp90
  • 05:52Data Analysis
  • 09:08Results: Protein Interactions of Hsp90
  • 10:47Conclusion

Summary

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Here, we present a protocol to obtain three-color smFRET data and its analysis with a 3D ensemble Hidden Markov Model. With this approach, scientists can extract kinetic information from complex protein systems, including cooperativity or correlated interactions.

Tags

Keywords: Three-color Single-molecule FRETProtein InteractionsCooperativityQuantitative BiologyProtein ComplexesDynamic InteractionsFlow Chamber AssemblyPEG-biotin Functionalized SlideMulti-color Prism TIRF MicroscopeEMCCD Camera Configuration

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