title: Iodixanol Gradient Centrifugation to Separate Components of the Low-Density Membrane Fraction from 3T3-L1 Adipocytes.
abstract: We optimized a set of fractionation techniques to facilitate the isolation of subcellular compartments containing insulin-sensitive glucose transporter isoform 4 (GLUT4), which is mobilized from GLUT4 storage vesicles (GSVs) in fat and muscle cells in response to insulin. In the absence of insulin, GLUT4 undergoes a continuous cycle of GSV formation and fusion with other compartments. Full membrane fractionation of 3T3-L1 adipocytes produces a low-density membrane fraction that contains both the constitutive recycling pool (the endosomal recycling compartments) and the insulin-sensitive pool (the GSVs). These two pools can be separated based on density using iodixanol gradient centrifugation, described here.
authors.affiliation: University of Glasgow, Glasgow G12 8QQ, United Kingdom;
authors.forename: Jessica B A
authors.initials: JB
authors.lastname: Sadler
authors.affiliation: Cancer Research UK, London Research Institute, London EC1V 4AD, United Kingdom;
authors.forename: Christopher A
authors.initials: CA
authors.lastname: Lamb
authors.affiliation: University of Glasgow, Glasgow G12 8QQ, United Kingdom;
authors.forename: Gwyn W
authors.initials: GW
authors.lastname: Gould
authors.affiliation: University of York, Heslington, York YO10 5DD, United Kingdom.
authors.forename: Nia J
authors.initials: NJ
authors.lastname: Bryant
mesh_terms: D041721:3T3-L1 Cells / Q000737:chemistry* / Q000502:physiology*; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods*; D008566:Membranes / Q000737:chemistry*; D051379:Mice; D014283:Triiodobenzoic Acids
chemical_list: D014283:Triiodobenzoic Acids; C044834:iodixanol