Isolation of GLUT4 Storage Vesicles

Konstantin V. Kandror1, Paul F. Pilch1

1 Boston University School of Medicine, Boston, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.20
DOI:  10.1002/0471143030.cb0320s30
Online Posting Date:  April, 2006
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Abstract

The immunoisolation of GLUT4‐containing vesicles from adipocytes is described in this unit. The methods involve homogenization of cells followed by differential centrifugation to provide the intracellular membranes that contain GLUT4. Subsequently, an immobilized monoclonal antibody is used for the isolation of vesicles of very high purity. The various protocols are applicable to cultured and primary adipocytes as well as skeletal muscle, the major insulin target cells expressing GLUT4.

Keywords: adipocytes; muscle; GLUT4; immunoadsorption; vesicle isolation

     
 
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Table of Contents

  • Basic Protocol 1: Immunoisolation of GLUT4 Vesicles From Rat Adipose Cells
  • Alternate Protocol 1: Immunoisolation of GLUT4 Vesicles from Cultured Adipose Cells
  • Alternate Protocol 2: Immunoisolation of GLUT4 Vesicles from Rat Skeletal Muscle
  • Alternate Protocol 3: Immunoisolation of GLUT4‐Containing Vesicles Using Magnetic Beads
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Immunoisolation of GLUT4 Vesicles From Rat Adipose Cells

  Materials
  • 1 mg/ml protein A–purified mouse monoclonal anti‐GLUT4 antibody (e.g., R & D Systems 1F8) in 0.1 M sodium borate, pH 8.6 (see James et al., )
  • 1 mg/ml protein A–purified nonspecific mouse IgG in 0.1 M sodium borate, pH 8.6
  • Reacti‐Gel (GF‐2000) beads (Pierce)
  • PBS ( appendix 2A)
  • PBS ( appendix 2A)/0.02% (w/v) NaN 3
  • Male Sprague‐Dawley rats, 150 to 175 g
  • KRH buffer (see recipe), 37°C
  • Type I collagenase specified for adipocyte isolation (Worthington) or equivalent
  • PBS with inhibitors (see recipe), 37°C, 18°C to 20°C, and 4°C
  • PBS ( appendix 2A)/1% (w/v) BSA
  • PBS ( appendix 2A)/1% (v/v) Triton X‐100
  • Laemmli SDS‐PAGE sample buffer without reducing agents (unit 6.1)
  • CO 2 chamber (or other equipment for IACUC‐approved protocols for sacrificing rats)
  • Dialysis tubing
  • 1.5‐ml microcentrifuge tubes or cryovials
  • −80°C freezer
  • Sharp scissors
  • 50‐ml plastic vials with tight caps
  • 37°C water bath with orbital and reciprocal shaker
  • Low‐speed medical centrifuge
  • 300‐µm nylon mesh
  • Long‐tip, 9‐in. Pasteur pipets
  • Vacuum pump with trap
  • 50‐ml centrifuge tube
  • Motor‐driven tissue grinder with a Teflon pestle (Potter‐Elvehjem type, Thomas model #3431, size B or C)
  • High‐speed centrifuge (e.g., Sorvall RC‐5C with SA‐600 rotor)
  • Gel‐loading tip (optional)
  • Spectrophotometer
  • 1.5‐ml microcentrifuge tubes
  • Tube rotator in a 4°C incubator
  • Additional reagents and equipment for protein quantification ( appendix 3B & ) and SDS‐PAGE (unit 6.1) or immunoprecipitation (unit 7.2) and immunoblotting (unit 6.2)

Alternate Protocol 1: Immunoisolation of GLUT4 Vesicles from Cultured Adipose Cells

  • 3T3‐L1 preadipocytes (ATTC CL‐173; Green and Kehinde, ) or other adipocyte cell lines (Gross et al., )
  • DMEM supplemented with 10% (v/v) bovine calf serum (DMEM‐10)
  • Differentiation medium (see recipe)
  • Maintenance medium (see recipe)
  • 1 mg/ml protein A–purified mouse monoclonal anti‐GLUT4 antibody (1F8) in 0.1 M sodium borate, pH 8.6
  • 1 mg/ml protein A–purified nonspecific mouse IgG in 0.1 M sodium borate, pH 8.6
  • Reacti‐Gel (GF‐2000) beads (Pierce)
  • PBS with inhibitors (see recipe)
  • Cell scraper
  • Ball‐bearing homogenizer (Isobiotec; Heidelberg, Germany) with a 12‐µm clearance.
  • Tube rotator
  • Additional reagents and equipment for tissue culture (unit 1.1)
NOTE: All solutions and equipment coming into contact with cells must be sterile, and proper aseptic technique should be used accordingly.NOTE: All culture incubations should be performed in a humidified 37°C, 5% CO 2 incubator unless otherwise specified. Some media, e.g., DMEM, require higher levels of CO 2 to maintain pH 7.4.

Alternate Protocol 2: Immunoisolation of GLUT4 Vesicles from Rat Skeletal Muscle

  Materials
  • Male Sprague‐Dawley rats, 150 to 175 g
  • PBS with inhibitors (see recipe)
  • 1 mg/ml protein A–purified anti‐GLUT4 antibody (e.g., R & D Systems 1F8) in 0.1 M sodium borate, pH 8.6
  • 1 mg/ml protein A–purified non‐specific mouse IgG in 0.1 M sodium borate, pH 8.6
  • Reacti‐Gel (GF‐2000) beads (Pierce)
  • 10% to 30% (w/w) linear sucrose gradients
  • Chamber with CO 2 or other equipment for IACUC‐approved protocol for sacrificing rats
  • Sharp scissors
  • Polytron homogenizer, 0.5 in. generator
  • 50‐ml centrifuge tubes
  • High‐speed centrifuge (e.g., Sorvall RC‐5C)
  • Ultracentrifuge with SW‐55Ti rotor and 5‐ml tubes, or comparable
  • Peristaltic pump
  • 1.5‐ml collection tubes

Alternate Protocol 3: Immunoisolation of GLUT4‐Containing Vesicles Using Magnetic Beads

  • Dynabead M‐280‐sheep anti‐mouse IgG (Dynal Biotech)
  • Magnetic bead separator (Dynal Biotech)
  • Tube rotator
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Figures

Videos

Literature Cited

Literature Cited
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