Isolation of Mitochondria-Associated ER Membranes (MAMs), Synaptic MAMs, and Glycosphingolipid Enriched Microdomains (GEMs) from Brain Tissues and Neuronal Cells.

Subcellular fractionation is a valuable procedure in cell biology to separate and purify various subcellular constituents from one another, i.e., nucleus, cytosol, membranes/organelles, and cytoskeleton. The procedure relies on the use of differential centrifugation of cell and tissue homogenates. Fractionated subcellular organelles may be subjected to additional purification steps that enable the isolation of specific cellular sub-compartments, including interorganellar membrane contact sites. Here we outline a protocol tailored to the isolation of mitochondria, mitochondria-associated ER membranes (MAMs), and glycosphingolipid enriched microdomains (GEMs) from the adult mouse brain, primary neurospheres, and murine embryonic fibroblasts (MEFs). We also provide a detailed protocol for the purification of synaptosomes and their corresponding MAMs .