title: Complete Membrane Fractionation of 3T3-L1 Adipocytes.
abstract: Fractionation techniques can facilitate the isolation of intracellular organelles containing insulin-sensitive glucose transporter isoform 4 (GLUT4), which is mobilized from GLUT4 storage vesicles in fat and muscle cells in response to insulin. This protocol for the full membrane fractionation of 3T3-L1 adipocytes results in five distinct fractions. A heavy membrane-containing pellet is produced and then further separated into the plasma membrane, mitochondrial and nuclear, and high-density membrane fractions. The initial supernatant is subjected to a series of centrifugation steps to isolate the low-density membrane fraction, which contains the majority of the insulin-sensitive pool of GLUT4; the supernatant produced in this step is the soluble fraction. The distribution of GLUT4 in fractions from insulin-stimulated versus untreated cells is assessed via immunoblotting.
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*; D008565:Membrane Proteins / Q000032:analysis; D008566:Membranes / Q000737:chemistry*; D051379:Mice
chemical_list: D008565:Membrane Proteins