title: 16K Fractionation of 3T3-L1 Adipocytes to Produce a Crude GLUT4-Containing Vesicle Fraction.
abstract: The insulin-sensitive pool of glucose transporter isoform 4 (GLUT4) can be isolated from total cell membranes using the 16K fractionation protocol, described here. This method produces a light membrane-containing supernatant that includes the insulin-sensitive pool of GLUT4 in GLUT4 storage vesicles. The 16K pellet fraction contains the heavy membranes (including the plasma membrane, mitochondria, nuclei, Golgi apparatus, and endoplasmic reticulum). The distribution of proteins between the two fractions is determined via immunoblotting. By subjecting insulin-stimulated versus unstimulated cells to this protocol, the mobilization of proteins out of the insulin-sensitive GLUT4 pool can be assessed.
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: D017667:Adipocytes / Q000648:ultrastructure*; D000818:Animals; D002458:Cell Fractionation / Q000379:methods*; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry*; D022162:Cytoplasmic Vesicles / Q000737:chemistry*; D051275:Glucose Transporter Type 4 / Q000302:isolation & purification*; D051379:Mice
chemical_list: D051275:Glucose Transporter Type 4; C495693:Slc2a4 protein, mouse