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Isolation of Synapse Sub-Domains by Subcellular Fractionation Using Sucrose Density Gradient Centrifugation: Purification of the Synaptosome, Synaptic Plasma Membrane, Postsynaptic Density, Synaptic Membrane Raft, and Postsynaptic Density Lattice

  • Tatsuo SuzukiEmail author
  • Yoshinori Shirai
  • Weidong Li
Protocol
Part of the Neuromethods book series (NM, volume 146)

Abstract

A protocol presents a purification of postsynaptic density (PSD), from rat brain by subcellular fractionation using solubilization of membrane with Triton X-100 and sucrose density centrifugation. The protocol also includes purification of other synapse sub-domains such as synaptosome, synaptic plasma membrane (SPM), synaptic membrane raft, PSD lattice, P1 (nuclei and cell debris), P2 (crude mitochondria fraction), S3 (soluble fraction), and P3 (microsomal fraction). The PSD purification method presented in this text is the one established by Siekevitz group. The PSDs obtained by this method are mainly excitatory type I PSDs. These methods are useful for biochemical analyses such as identification of proteins associated with these sub-domains by proteomics methods and western blotting, and morphological analyses at the electron microscopic level. The purification protocol for the synaptic membrane raft using sucrose gradient ultracentrifugation is a useful means by which to analyze the relationship between the PSD and synaptic membrane raft by isolating both preparations simultaneously.

Keywords

Synaptosome Synaptic plasma membrane Postsynaptic density Postsynaptic membrane raft PSD lattice Subcellular fractionation Detergent-insoluble cytoskeleton Detergent-insoluble membrane 

Notes

Acknowledgments

The author learned the method of PSD purification in the Philip Siekevitz laboratory, Rockefeller University, New York. The author heartily thanks Dr. Philip Siekevitz and Marie LeDoux for their instruction.

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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Department of Molecular and Cellular Physiology, Institute of MedicineShinshu University Academic AssemblyMatsumotoJapan
  2. 2.Bio-X Institutes, Key Laboratory for the Genetics of Development and Neuropsychiatric Disorders (Ministry of Education), Shanghai Key Laboratory of Psychotic Disorders, Brain Science and Technology Research Center, Institute of psychology and Behavioral SciencesShanghai Jiao Tong UniversityShanghaiChina
  3. 3.Institute for Biomedical Sciences, Interdisciplinary Cluster for Cutting Edge ResearchShinshu UniversityMatsumotoJapan

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