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

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.