Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation

John M. Graham1

1 Liverpool John Moores University, Liverpool
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.9
DOI:  10.1002/0471143030.cb0309s10
Online Posting Date:  May, 2001
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Abstract

This unit describes the isolation of Golgi membranes on a preparative basis. Methods are provided for rapid isolation of dextran‐treated Golgi stacks from rat liver using a sucrose density barrier, and Golgi isolation by floatation from a light mitochondrial fraction, along with an alternate procedure using a self‐generated iodixinol gradient. If the Golgi tends to vesiculate during homogenization (commonly the case with cultured cells), a primary requirement is to separate these vesicles from other microsomal compartments, so a procedure is described for a discontinuous gradient of sucrose for cultured cells, and an alternate protocol describes a continuous iodixinol gradient that may provide greater resolution. A self‐generated iodixinol gradient is described to prepare Golgi membranes from a microsomal fraction of rat hepatocytes, and a standard Golgi enzyme marker assay is given in a support protocol.

     
 
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Table of Contents

  • Basic Protocol 1: Rapid Isolation of Golgi Membranes from Rat Liver Using a Sucrose Density Barrier
  • Basic Protocol 2: Isolation of Golgi Membranes from a Rat Liver Light Mitochondrial Fraction by Flotation Through a Discontinuous Sucrose Gradient
  • Alternate Protocol 1: Isolation of Golgi Membranes from a Rat Liver Light Mitochondrial Fraction in a Self‐Generated Gradient of Iodixanol
  • Basic Protocol 3: Isolation of Golgi Membranes from Cultured Cells by Flotations Through a Discontinuous Sucrose Gradient
  • Alternate Protocol 2: Isolation of Golgi Membranes from Cultured Cells in a Continuous Gradient of Iodixanol
  • Basic Protocol 4: Isolation of Golgi Membranes from a Microsomal Fraction of Hepatocytes in a Self‐Generated Gradient of Iodixanol
  • Support Protocol 1: Assay for UDP‐Galactose Galactosyltransferase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Rapid Isolation of Golgi Membranes from Rat Liver Using a Sucrose Density Barrier

  Materials
  • 150‐ to 200‐g Sprague‐Dawley rat
  • DHM (see recipe)
  • 1.2 M sucrose density barrier (see recipe)
  • HM (see recipe), optional
  • Crude α‐amylase Type X‐A from Aspergillus oryzae (Sigma Aldrich Co.), optional
  • α‐Amylase Type VIII‐A from barley (Sigma Aldrich Co.), optional
  • Dissecting tools, including scissors, razor blade, and forceps
  • Polytron homogenizer (Brinkmann Instruments)
  • Phase‐contrast microscope
  • High‐speed centrifuge with swinging‐bucket rotor and 30‐ to 50‐ml clear plastic tubes
  • 10‐ml syringes with long metal cannulas (i.d. ∼1 mm) or Pasteur pipet attached to an aspirator
  • Glass rod
  • Ultracentrifuge with swinging‐bucket rotor (Beckman SW 28.1, Sorvall AH629, or equivalent) and 17‐ml tubes
  • Additional reagents and equipment for determining protein concentration ( appendix 3B)
NOTE: Protease inhibitors (unit 3.4) can be added to any or all solutions.

Basic Protocol 2: Isolation of Golgi Membranes from a Rat Liver Light Mitochondrial Fraction by Flotation Through a Discontinuous Sucrose Gradient

  Materials
  • 150‐ to 200‐g Sprague‐Dawley rat
  • HM (see recipe)
  • 2.0, 1.33, 1.2, 1.1, 0.77, and 0.25 M sucrose gradient solutions (see recipe)
  • Dissecting tools, including scissors or razor blade
  • Potter‐Elvehjem homogenizer, ∼0.09‐mm clearance, 40‐ml working volume (Fisher Scientific), with chilled pestle
  • Overhead high‐torque electric motor, thyristor controlled (Fisher Scientific)
  • Low‐speed centrifuge with swinging‐bucket rotor and 50‐ml tubes
  • ∼5‐ and ∼30‐ml Dounce homogenizers (Wheaton) with loose‐fitting type‐B conical pestles
  • High‐speed centrifuge with 8 × 50–ml fixed‐angle rotor (e.g., Sorvall SS34)
  • 10‐ml syringe and metal cannula
  • Refractometer
  • Ultracentrifuge with swinging‐bucket rotor (Beckman SW 28.1, Sorvall AH629, or equivalent) and 17‐ml tubes
  • Additional reagents and equipment for determining protein concentration ( appendix 3B)
NOTE: Protease inhibitors (unit 3.4) can be added to any or all solutions.

Alternate Protocol 1: Isolation of Golgi Membranes from a Rat Liver Light Mitochondrial Fraction in a Self‐Generated Gradient of Iodixanol

  • HME (see recipe), optional
  • OptiPrep
  • ODB solution (see recipe)
  • IWS solution (see recipe)
  • High‐density cushion (20%, w/v, iodixanol): 2:3 (v/v) recipeIWS/ recipeHME (or recipeHM)
  • Ultracentrifuge with vertical (e.g., Beckman VTi 65.1, Sorvall 65V13), near‐vertical (e.g., Beckman NVT 65) rotor and ∼11‐ml sealable ultracentrifuge tubes or low‐angle fixed‐angle rotor (∼20°; e.g., Beckman 50.3Ti) and ∼5‐ml sealable ultracentrifuge tubes.
NOTE: Protease inhibitors (unit 3.4) can be added to any or all solutions.

Basic Protocol 3: Isolation of Golgi Membranes from Cultured Cells by Flotations Through a Discontinuous Sucrose Gradient

  Materials
  • Cultured cells
  • HM (see recipe)
  • 0.8, 1.2, 1.6, and 2.0 M sucrose gradient solutions (see recipe)
  • Ball‐bearing homogenizer
  • 5‐ml syringe and metal cannula
  • Ultracentrifuge with swinging‐bucket rotor (Beckman SW 28.1, Sorvall AH629, or equivalent) and ∼17‐ml tubes, 4°C
  • Additional reagents and equipment for determining protein concentration ( appendix 3B)
NOTE: Protease inhibitors (unit 3.4) can be added to any or all solutions.

Alternate Protocol 2: Isolation of Golgi Membranes from Cultured Cells in a Continuous Gradient of Iodixanol

  • Cellular homogenate (see protocol 4, step )
  • 25% (w/v) iodixanol: 1:1 (v/v) IWS solution (see recipe) and HM (see recipe)
  • Low‐speed centrifuge with swinging‐bucket rotor and 10‐ to 15‐ml tubes, all at 4°C
  • Ultracentrifuge with fixed‐angle rotor and 5‐ to 10‐ml tubes (optional), all at 4°C
  • 5‐ to 10‐ml Dounce homogenizer (Wheaton) with loose‐fitting type‐B pestle (optional)
  • Two‐chamber gradient maker or Gradient Master (e.g., BioComp Instruments)
  • Gradient unloader (upward displacement; Axis‐Shield)
  • Refractometer
  • Additional reagents and equipment for determining protein concentration ( appendix 3B)
NOTE: Protease inhibitors (unit 3.4) can be added to any or all solutions.

Basic Protocol 4: Isolation of Golgi Membranes from a Microsomal Fraction of Hepatocytes in a Self‐Generated Gradient of Iodixanol

  Materials
  • 95% O 2/5% CO 2 gas mixture
  • DMEM/BSA: Dulbecco's modified Eagle medium ( appendix 2A) with 1.0% (w/v) BSA
  • Collagenase‐prepared rat hepatocytes (Plonné et al., )
  • PBS ( appendix 2A), 4°C
  • 10 mM HEPES‐NaOH buffer, pH 7.8
  • HB (see recipe)
  • SB (see recipe)
  • OptiPrep (Axis‐Shield, Life Technologies, Accurate Chemicals)
  • 30% (w/v) iodixanol: 1:1 (v/v) OptiPrep/ recipeSB
  • 15% (w/v) iodixanol: 1:3 (v/v) OptiPrep/ recipeSB
  • Low‐speed centrifuge with swinging‐bucket rotor and 50‐ml conical tubes, all at 4°C
  • ∼10‐ml Dounce homogenizer (Wheaton) with tight‐fitting type‐A pestle
  • High‐speed centrifuge with fixed‐angle rotor and ∼15‐ml tubes (e.g., Sorvall SE12), 4°C
  • Ultracentrifuge with the following, all at 4°C:
  •  Fixed‐angle rotor (e.g., Beckman 50Ti, SorvallT865.1) and ∼10‐ml tubes
  •  Vertical rotor (e.g., Beckman VTi 65.1, Sorvall 65V13), near‐vertical rotor (e.g., Beckman NVT 65), with ∼11‐ml sealable tubes or a low‐angle fixed‐angle rotor (∼20°; e.g., Beckman 50.3Ti) and ∼5‐ml sealable ultrcentrifuge tubes.
  •  ∼10‐ml Dounce homogenizer (Wheaton) with loose‐fitting type‐B pestle
  • Gradient unloader (upward displacement; e.g., Axis‐Shield)
  • Additional reagents and equipment for determining protein concentration ( appendix 3B)
NOTE: Protease inhibitors (unit 3.4) can be added to any or all solutions.

Support Protocol 1: Assay for UDP‐Galactose Galactosyltransferase

  Materials
  • 10% (w/v) trichloroacetic acid (TCA)
  • Acceptor solution (see recipe)
  • UDP‐gal solution (see recipe)
  • 2.4‐cm filter paper discs
  • Polystyrene board
  • Pins
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Figures

Videos

Literature Cited

Literature Cited
   Axis‐Shield. 2000. Types of centrifugal separations. In Axis‐Shield Density Gradient Media Catalog, pp. 13‐29. Axis‐Shield, Oslo, Norway; Life Technologies, Grand Island, N.Y.; Accurate Chemicals, Westbury, N.Y.
   Balch, W.E. and Rothman, J.E. 1985. Characterization of protein transport between successive compartments of the Golgi apparatus: Asymmetric properties of donor and acceptor activities in cell‐free systems. Arch. Biochem. Biophys. 240:413‐425.
   Balch, W.E., Dunphy, W.G., Braell, W.A., and Rothman, J.E. 1984. Reconstitution of the transport of protein between successive compartments of the Golgi measured by the coupled incorporation of N‐acetylglucosamine. Cell 39:405‐416.
   Beaufay, H., Amar‐Costesec, A., Feytmans, E., Thines‐Sempoux, D., Wibo, M., Robbi, M., and Berthet, J. 1974. Analytical study of microsomes and isolated subcellular fractions from rat liver. J. Cell Biol. 61:188‐200.
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   Cartwright, I.J., Higgins, J.A., Wilkinson, J., Bellavia, S., Kendrick, J.S., and Graham, J.M. 1997. Investigation of the role of lipids in the assembly of very low density lipoproteins in rabbit hepatocytes. J. Lipid Res. 38:531‐545.
   Ehrenreich, J.H., Bergeron, J.J.M., Siekevitz, P., and Palade, G.E. 1973. Golgi fractions prepared from rat liver homogenates. J.Cell Biol. 59:45‐72.
   Evans, W.H. 1992. Isolation and characterization of membranes and cell organelles. In Preparative Centrifugation—A Practical Approach (D. Rickwood, ed.) pp. 233‐270. Oxford University Press, Oxford.
   Fleischer, B. and Fleischer, S. 1970. Preparation and characterization of Golgi membranes from rat liver. Biochim. Biophys. Acta 219:301‐319.
   Fleischer, B., Fleischer, S., and Ozawa, H. 1969. Isolation and characterization of Golgi membranes from bovine liver. J. Cell Biol. 43:59‐79.
   Ford, T., Rickwood, D., and Graham, J. 1983. Buoyant densities of macromolecules, macromolecular complexes and cell organelles in Nycodenz gradients. Anal. Biochem. 128:232‐239.
   Ford, T., Graham, J., and Rickwood, D. 1994. Iodixanol: A nonionic iso‐osmotic centrifugation medium for the formation of self‐generated gradients. Anal. Biochem. 220:360‐366.
   Graham, J.M. 1997. Homogenization of cells and tissues. In Subcellular Fractionation—A Practical Approach (J.M. Graham and D. Rickwood, eds.) pp. 1‐29. Oxford University Press, Oxford.
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   Plonné, D., Cartwright, I., Linss, W., Dargel, R., Graham, J.M., and Higgins, J.A. 1999. Separation of the intracellular secretory compartment of rat liver and isolated rat hepatocytes in a single step using self‐generating gradients of iodixanol. Anal. Biochem. 276:88‐96.
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