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

Article DOI: 10.3791/54127-v • 11:19 min • July 4th, 2016
July 4th, 2016 •
1, 1, 1,2
1Department of Physiology and Biophysics, Case Western Reserve University, 2School of Medicine, Case Western Reserve University

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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.

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Site Directed Spin Labeling EPR Spectroscopic Studies Pentameric Ligand-Gated Ion Channels Purification Reconstitution Electron Paramagnetic Resonance Studies Membrane Protein Dynamics Physiologically Relevant Environment Site Directed Mutagenesis GLIC Gene Cysteine Mutant Potassium Phosphate Buffer Autoclave Terrific Broth Media Glucose Kanamycin Glycerol Optical Density IPTG
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