Isolation of Peroxisomes 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 3.5
DOI:  10.1002/0471143030.cb0305s06
Online Posting Date:  May, 2001
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Abstract

Peroxisome purification depends on a two‐step procedure: differential centrifugation to prepare a light mitochondrial fraction and fractionation on a density‐gradient medium preferably iodixanol or Nycodenz, to isolate the peroxisome enriched fraction. The iodixanol gradient may be a preformed continuous gradient or a self‐generating gradient. Alternatively a continuous Nycodenz gradient or a simple Nycodenz barrier may be used for the second step. The unit contains protocols for peroxisome isolation from rat liver, tissue culture cells (HepG2 cells), and yeast spheroplasts. The extent of endoplasmic reticulum contamination of the prep can be assessed using an assay for the marker enzyme NADPH‐cytochrome creductase.

     
 
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Table of Contents

  • Basic Protocol 1: Isolation of a Light Mitochondrial Fraction from Rat Liver
  • Basic Protocol 2: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction Using a Preformed Continuous Iodixanol Gradient
  • Alternate Protocol 1: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction in a Self‐Generated Iodixanol Gradient
  • Basic Protocol 3: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction Using a Performed Continuous Nycodenz Gradient
  • Alternate Protocol 2: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction Using a Nycodenz Barrier
  • Basic Protocol 4: Isolation of Peroxisomes from Yeast Spheroplasts Using a Preformed Continuous Nycodenz Gradient
  • Basic Protocol 5: Isolation of Peroxisomes from Cultured Cells (HepG2) Using a Preformed Continuous Nycodenz Gradient
  • Support Protocol 1: Assay for Endoplasmic Reticulum Marker Enzyme NADPH‐Cytochrome c Reductase
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Isolation of a Light Mitochondrial Fraction from Rat Liver

  Materials
  • 150‐ to 200‐g Sprague‐Dawley rats
  • Homogenization medium (HM; see recipe)
  • Protease inhibitors (optional; see recipe) added to any or all solutions at concentrations indicated in recipe
  • Dissecting equipment
  • Overhead high‐torque electric motor (thyristor‐controlled) for homogenizers
  • Potter‐Elvehjem homogenizer (clearance ∼0.09 mm), 25‐ml working volume, prechilled
  • Dounce homogenizer (loose‐fitting Wheaton type B pestle, ∼30 ml), prechilled
  • Dounce homogenizer (loose‐fitting Wheaton type B pestle, ∼5 ml), prechilled
  • Low‐speed refrigerated centrifuge with swinging‐bucket rotor accommodating 50‐ml tubes
  • 40‐ to 50‐ml polycarbonate centrifuge tubes
  • High‐speed refrigerated centrifuge with fixed‐angle rotors accommodating 50‐ml tubes (e.g., Sorvall SS‐34)
NOTE: All solutions, glassware, centrifuge tubes and equipment should be pre‐cooled to 0° to 4°C and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature. When handling the glass vessel of the Potter‐Elvehjem homogenizer, a thermally insulated glove or silicone rubber hand protector should be used, not only to avoid heat transfer from the skin but also to protect the hand in the unlikely event of breakage of the vessel.

Basic Protocol 2: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction Using a Preformed Continuous Iodixanol Gradient

  Materials
  • Iodixanol gradient solutions A, B, and C (see recipe)
  • Light mitochondrial pellet (LMP) suspension (see protocol 1)
  • Homogenization medium (HM; see recipe)
  • Protease inhibitors (optional; see recipe) added to any or all solutions at concentrations indicated in recipe
  • Gradient maker: two‐chamber or Gradient Master (Accurate Chemical)
  • 5‐ml syringe with metal cannula (1‐mm i.d.).
  • Ultracentrifuge and thick‐walled polycarbonate tubes (30‐ml) for a fixed‐angle rotor (e.g., Beckman 60 Ti or Sorvall T‐865)
  • Gradient unloader for dense‐end‐first collection (thin metal cannula connected to a peristaltic pump)
  • Additional reagents and equipment for protein assay ( appendix 3B)
NOTE: All solutions, glassware, centrifuge tubes and equipment should be precooled to 0° to 4°C and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.

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

  • OptiPrep (60%, w/v iodixanol; Life Technologies, Accurate Chemical, Sigma, or Aldrich)
  • Diluent medium (DM; see recipe)
  • Vertical (e.g., Beckman VTi 65.1 or Sorvall 65V13), near‐vertical (e.g., Beckman NVT 65), or low‐angle (20° to 26°) fixed‐angle rotor for an ultracentrifuge with 11‐ to 12‐ml sealed tubes
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.

Basic Protocol 3: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction Using a Performed Continuous Nycodenz Gradient

  Materials
  • 20% (w/v) Nycodenz in homogenization medium (HM; see recipe)
  • 50% and 60% (w/v) Nycodenz in high‐density diluent (HD; see recipe)
  • Light mitochondrial pellet (LMP) suspension (see protocol 1)
  • Homogenization medium (HM; see recipe)
  • Protease inhibitors (optional; see recipe) added to any or all of the solutions at concentrations indicated in recipe
  • Gradient maker: two‐chamber or Gradient Master (Accurate Chemical)
  • 5‐ml syringe with metal cannula (1‐mm i.d.)
  • Ultracentrifuge with vertical rotor (e.g., Beckman VTi50 or Sorvall 50V39) with appropriate sealed tubes (∼39 ml)
  • Gradient unloader for dense‐end‐first collection (tube‐puncturing device or thin metal cannula connected to a peristaltic pump)
  • Additional reagents and equipment for protein assay ( appendix 3B)
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.

Alternate Protocol 2: Isolation of Peroxisomes from a Rat Liver Light Mitochondrial Fraction Using a Nycodenz Barrier

  • 30% (w/v) Nycodenz in high‐density diluent (HD; see recipe)
  • Fixed‐angle rotor with ∼25‐ml thick‐walled polycarbonate tubes (e.g., Beckman 55.2 Ti or Sorvall T‐865)
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.

Basic Protocol 4: Isolation of Peroxisomes from Yeast Spheroplasts Using a Preformed Continuous Nycodenz Gradient

  Materials
  • Yeast homogenization medium (YHM; see recipe)
  • 15% (w/v) Nycodenz in yeast low‐density diluent (YLD; see recipe)
  • 42.5% and 50% (w/v) Nycodenz in yeast high‐density diluent (YHD; see recipe)
  • Yeast low‐density diluent (YLD; see recipe)
  • Yeast high‐density diluent (YHD; see recipe)
  • Protease inhibitors (optional; see recipe) added to any or all solutions at concentrations indicated in recipe
  • Dounce homogenizer (Wheaton Type B, 40‐ml)
  • Dounce homogenizer (Wheaton Type B, 5‐ to 10‐ml)
  • Gradient maker: two‐chamber or Gradient Master (Accurate Chemical)
  • 5‐ml syringe with metal cannula (1‐mm i.d.)
  • Ultracentrifuge with vertical rotor (e.g., Beckman VTi65.1 or Sorvall 65V13) with appropriate sealable tubes (∼13 ml)
  • Gradient unloader for dense‐end‐first collection (tube‐puncturing device or thin metal cannula connected to a peristaltic pump)
  • High‐speed refrigerated centrifuge with fixed‐angle rotor to accommodate 50‐ml tubes (e.g., Sorvall SS34)
  • Additional reagents and equipment for preparation of yeast spheroplasts (unit 3.3) and protein assay ( appendix 3B)
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.

Basic Protocol 5: Isolation of Peroxisomes from Cultured Cells (HepG2) Using a Preformed Continuous Nycodenz Gradient

  Materials
  • Suspension of cells (∼108 cells) in 3 to 5 ml of homogenization medium (HM; see recipe)
  • Homogenization medium (HM; see recipe)
  • 10% (w/v) Nycodenz in homogenization medium (HM; see recipe)
  • 40% and 50% (w/v) Nycodenz in high‐density diluent (HD; see recipe)
  • Protease inhibitors (optional; see recipe) added to any or all solutions at concentrations indicated in recipe
  • Ball‐bearing homogenizer (see Balch and Rothman, , for details of construction)
  • Low‐speed centrifuge with swinging‐bucket rotor to accommodate 10‐ to 20‐ml tubes
  • Dounce homogenizer (5‐ to 10‐ml, Wheaton type B)
  • High‐speed centrifuge with fixed‐angle rotor to accommodate 10‐ to 14‐ml tubes (e.g., Sorvall SE12)
  • Gradient maker: two‐chamber or Gradient Master (Accurate Chemical)
  • 5‐ml syringe with metal cannula (1‐mm i.d.)
  • Ultracentrifuge with vertical rotor (e.g., Beckman VTi65.1or Sorvall 65V13) with appropriate sealable tubes (∼13‐ml)
  • Gradient unloader for dense‐end‐first collection (tube‐puncturing device or thin metal cannula connected to a peristaltic pump)
  • Additional reagents and equipment for protein assay ( appendix 3B)
NOTE: All solutions, glassware, centrifuge tubes, and equipment should be precooled to 0° to 4°C and kept on ice throughout. Centrifuge rotors should be precooled to the same temperature.

Support Protocol 1: Assay for Endoplasmic Reticulum Marker Enzyme NADPH‐Cytochrome c Reductase

  Materials
  • Assay buffer (see recipe)
  • 25 mg/ml cytochrome c in recipeassay buffer (prepare fresh; keep on ice)
  • 10 mM EDTA (dilute 1 ml 100 mM EDTA stock to 10 ml with H 2O)
  • 1 mg/ml rotenone in ethanol (store up to 1 month at 4°C)
  • 2 mg/ml NADPH in recipeassay buffer (prepare fresh; keep on ice away from light)
  • Recording spectrophotometer (visible wavelength) with 1‐ml cuvettes
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Figures

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Literature Cited

Literature Cited
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