Summary
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.
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Acknowledgements
This work was supported by in part by the National Institutes of Health, NIDDK grants KO1-DK62264, RO1-DK26523, RO1-DK61765, PO1-DK44484, PO1-DK72084, and R24-DK64388 (Hopkins Digestive Diseases Basic Research Development Core Center); Broad Medical Research Program grant (IBD-0119R2); and the Hopkins Center for Epithelial Disorders. We thank Elsevier Inc. for permission to reuse the figures originally published in its journal Gastroenterology.
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Li, X., Donowitz, M. (2008). Fractionation of Subcellular Membrane Vesicles of Epithelial and Nonepithelial Cells by OptiPrep™ Density Gradient Ultracentrifugation. In: Ivanov, A.I. (eds) Exocytosis and Endocytosis. Methods in Molecular Biology, vol 440. Humana Press. https://doi.org/10.1007/978-1-59745-178-9_8
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DOI: https://doi.org/10.1007/978-1-59745-178-9_8
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