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Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)
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JoVE Journal Biologia
Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

DOI:
10.3791/52509-v

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08:14 min

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April 20, 2015

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, ,
1Department of Physiology and Biophysics,Chicago Medical School

Capitoli

  • 00:05Titolo
  • 01:42Transfection of Cells and Cell Culture
  • 03:02Establishing Optimal Conditions for Plasma Membrane Permeabilization
  • 04:59Protease Treatment of Fluorescent Proteins and Image Analysis
  • 05:56Results: Analysis of Protein Topology Using FPP
  • 07:48Conclusion

Summary

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Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

Tags

Membrane Protein TopologyFluorescence Protease Protection (FPP)GFP TaggingDigitonin PermeabilizationProtease DegradationSubcellular OrganellesProtein Orientation

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