Fractionation of subcellular membrane vesicles of epithelial and nonepithelial cells by OptiPrep density gradient ultracentrifugation.
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, a few years ago synthetic OptiPrep (iodixanol) began being used for separation of organelles because of 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.
Li
Xuhang
X
GI Division, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, MD, USA.
Donowitz
Mark
M
D000818:Animals; D015153:Blotting, Western; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002499:Centrifugation, Density Gradient; D006224:Cricetinae; D004591:Electrophoresis, Polyacrylamide Gel; D004847:Epithelial Cells / Q000648:ultrastructure*; D006801:Humans; D007413:Intestinal Mucosa / Q000648:ultrastructure; D007425:Intracellular Membranes / Q000648:ultrastructure*; D021962:Membrane Microdomains / Q000000981:diagnostic imaging; D008871:Microvilli / Q000648:ultrastructure; D015388:Organelles / Q000648:ultrastructure*; D011817:Rabbits; D014283:Triiodobenzoic Acids; D014463:Ultrasonography
D014283:Triiodobenzoic Acids; C044834:iodixanol