Density gradient ultracentrifugation (DGUC) is widely used for physical isolation (enrichment rather than purifi cation) of subcellular membrane vesicles. It has been a valuable tool to study specifi c subcellular localization and dynamic traffi cking 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.
- Density gradient ultracentrifugation
- Lipid rafts
- Subcellular membrane vesicles
ASJC Scopus subject areas
- Molecular Biology