title: Fractionation of subcellular membrane vesicles of epithelial and non-epithelial cells by OptiPrep™ density gradient ultracentrifugation.
abstract: Density gradient ultracentrifugation (DGUC) is widely used for physical isolation (enrichment rather than purification) of subcellular membrane vesicles. It has been a valuable tool to study specific subcellular localization and dynamic trafficking of proteins. While sucrose has been the main component of density gradients, several years ago, synthetic OptiPrep™ (iodixanol) began being used for separation of organelles due to its iso-osmotic property. Here, we describe a detailed protocol for density gradient fractionation of various mammalian subcellular vesicles, including endoplasmic reticulum (ER), Golgi apparatus, endosomes, and lipid rafts, as well as apical and basolateral membranes of polarized epithelial cells.
authors.affiliation: GI Division, Department of Medicine, Johns Hopkins University School of Medicine, 918 Ross Research Bldg, 720 Rutland Ave, 21205, Baltimore, MD, USA, xuhang@jhmi.edu.
authors.forename: Xuhang
authors.initials: X
authors.lastname: Li
authors.forename: Mark
authors.initials: M
authors.lastname: Donowitz
mesh_terms: D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002499:Centrifugation, Density Gradient / Q000379:methods; D022162:Cytoplasmic Vesicles / Q000737:chemistry*; D004847:Epithelial Cells / Q000737:chemistry / Q000378:metabolism*; D006801:Humans; D013347:Subcellular Fractions