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Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels
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JoVE Journal Chimica
Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

Site Directed Spin Labeling and EPR Spectroscopic Studies of Pentameric Ligand-Gated Ion Channels

DOI:
10.3791/54127-v

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11:19 min

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July 04, 2016

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, ,
1Department of Physiology and Biophysics,Case Western Reserve University, 2School of Medicine,Case Western Reserve University

Capitoli

  • 00:05Titolo
  • 00:51Gloeobacter violaceus (GLIC) Expression and Purification
  • 05:31GLIC Reconstitution in Asolectin Membranes
  • 07:33EPR Measurements
  • 08:42Results: Characterization of Pentameric Ligand-Gated Ion Channels
  • 10:06Conclusion

Summary

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This article describes methods for site-directed spin labeling and reconstitution of pentameric ligand-gated channels for Electron Paramagnetic Resonance studies. This protocol can be adapted for any membrane protein. The reconstitution method described here can also be used for patch-clamp measurements of macroscopic and single-channel currents in a defined lipid system.

Tags

Keywords: Site Directed Spin LabelingEPR SpectroscopyPentameric Ligand-gated Ion ChannelsMembrane Protein DynamicsGLICSite-directed MutagenesisProtein PurificationCell LysisMembrane Protein Reconstitution

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