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

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.