Isolation of Synapse Subdomains by Subcellular Fractionation Using Sucrose Density Gradient Centrifugation

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 subdomains such as synaptosome, synaptic plasma membrane, P₁ (nuclei and cell debris), P₂ (crude mitochondria fraction), S₃ (soluble fraction), and P₃ (microsomal fraction). The method presented in this text is the one established by Siekevitz group. The PSDs obtained by this method are mainly excitatory type I PSDs. The method has been widely used and is useful for biochemical analyses such as identification of proteins associated with these subdomains by proteomics methods and western blotting, and morphological analyses at the electron microscopic level.