Purification of a Crude Mitochondrial Fraction by Density‐Gradient Centrifugation

John M. Graham1

1 Liverpool John Moores University, Liverpool
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.4
DOI:  10.1002/0471143030.cb0304s04
Online Posting Date:  May, 2001
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Abstract

Most mitochondria prepared by differential centrifugation are contaminated to some extent by lysosomes, peroxisomes, tubular Golgi membranes, and small amounts of endoplasmic reticulum. Density gradient centrifugation using a variety of density media‐‐sucrose, Percoll, Nycodenz, Iodixanol‐‐is described here and is used to prepare purer fractions of mitochondria. The resulting gradient can be analyzed for three marker enzymes: succinate dehydrogenase (mitochondria), b‐galactosidase (lysosomes), and catalase (peroxisomes).

     
 
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Table of Contents

  • Basic Protocol 1: Resolution of a Rat Liver Mitochondrial Fraction in a Continuous Sucrose Gradient
  • Basic Protocol 2: Isolation of Mitochondria from Rat Brain Using a Discontinuous Percoll Gradient
  • Alternate Protocol 1: Isolation of Mitochondria from Rat Liver Using a Discontinuous Percoll Gradient
  • Basic Protocol 3: Resolution of Mitochondrial Fraction in a Self‐Generated Percoll Gradient
  • Basic Protocol 4: Resolution of a Rat Liver Mitochondrial Fraction in an Iodixanol Gradient
  • Support Protocol 1: Succinate Dehydrogenase Assay for Mitochondria
  • Support Protocol 2: β‐Galactosidase Assay for Lysosomes
  • Support Protocol 3: Catalase Assay for Peroxisomes
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Resolution of a Rat Liver Mitochondrial Fraction in a Continuous Sucrose Gradient

  Materials
  • General‐purpose homogenization medium (GHM; see recipe), ice cold
  • Sucrose gradient solutions (see recipe)
  • Sucrose cushion (see recipe)
  • Protease inhibitors (optional; see recipe)
  • Dounce homogenizer (5‐ to 10‐ml with loose‐fitting, Wheaton type B pestle)
  • Low‐speed centrifuge with swinging‐bucket rotor and appropriate tubes
  • High‐speed centrifuge with fixed‐angle rotor and appropriate tubes
  • Gradient maker (two‐chamber or Gradient Master)
  • Ultracentrifuge (e.g., Beckman VTi 50 with vertical rotor) and 39‐ml tubes
  • Gradient unloader (optional)
  • Additional reagents and equipment for preparing homogenates (unit 3.3)

Basic Protocol 2: Isolation of Mitochondria from Rat Brain Using a Discontinuous Percoll Gradient

  Materials
  • 150‐ to 200‐g Sprague‐Dawley rats
  • Brain homogenization medium (BHM; see recipe), ice cold
  • Percoll solutions in BHM: 15% (v/v), 23% (v/v), and 40% (v/v) (see recipe)
  • Protease inhibitors (optional; see recipe)
  • 10‐ml Dounce homogenizer with tight‐fitting and loose‐fitting pestles (Wheaton type A and B, respectively)
  • Low‐speed centrifuge with swinging‐bucket rotor and appropriate tubes
  • High‐speed centrifuge with fixed‐angle rotor and appropriate tubes
  • Ultracentrifuge with swinging‐bucket rotor for 12‐ml tubes

Alternate Protocol 1: Isolation of Mitochondria from Rat Liver Using a Discontinuous Percoll Gradient

  Materials
  • 150‐ to 200‐g Sprague‐Dawley rats
  • Mannitol‐sucrose homogenization medium (MSHM; see recipe), ice‐cold
  • Percoll solutions in MSHM: 19%, 31%, 42%, and 52% (v/v) (see recipe)
  • Protease inhibitors (optional; see recipe) can be added to any or all of the solutions
  • Low‐speed centrifuge with swinging‐bucket rotor and appropriate tubes
  • High‐speed centrifuge with fixed‐angle rotor (e.g., Sorvall SS34) with ∼14‐ml tubes
  • Additional reagents and equipment for preparing liver homogenate (unit 3.3)

Basic Protocol 3: Resolution of Mitochondrial Fraction in a Self‐Generated Percoll Gradient

  Materials
  • Mannitol buffer A (see recipe), ice cold
  • 30% (v/v) Percoll solution (see recipe), ice cold
  • Protease inhibitors (optional; see recipe)
  • Low‐speed centrifuge with swinging‐bucket rotor and appropriate tubes (∼30 ml)
  • 5‐ and 30‐ml Dounce homogenizers with loose‐fitting pestles (Wheaton type B)
  • High‐speed centrifuge with fixed‐angle rotor and appropriate tubes (∼30 ml)
  • Ultracentrifuge with fixed‐angle rotor (e.g., Beckman 60 Ti or Sorvall T‐1250) and appropriate tubes
  • Additional reagents and equipment for preparing homogenates (unit 3.3)

Basic Protocol 4: Resolution of a Rat Liver Mitochondrial Fraction in an Iodixanol Gradient

  Materials
  • Iodixanol buffer A (see recipe), ice cold
  • 50% (w/v) iodixanol solution: 1 vol iodixanol buffer B (see recipe) in 5 vol OptiPrep (60% iodixanol; Nycomed Pharm, Life Technologies, Accurate Chemicals, Mediatech)
  • 19% and 27% iodixanol solutions: dilute 50% iodixanol with iodixanol buffer A
  • Protease inhibitors (optional; see recipe)
  • Low‐speed centrifuge with swinging‐bucket rotor and appropriate tubes
  • 5‐ and 20‐ml syringes and metal cannulae (i.d. ∼0.7 mm)
  • 5‐ and 30‐ml Dounce homogenizers with loose‐fitting pestles (Wheaton type B)
  • High‐speed centrifuge with fixed‐angle rotor and appropriate tubes
  • Ultracentrifuge with swinging‐bucket rotor (e.g., Beckman SW 28.1 or Sorvall AH‐629) and appropriate tubes
  • Refractometer (optional)
  • Gradient maker: two‐chamber or Gradient Master
  • Additional reagents and equipment for preparing homogenates (unit 3.3)

Support Protocol 1: Succinate Dehydrogenase Assay for Mitochondria

  Materials
  • Mitochondrial gradient fraction (see Basic Protocols protocol 11 to protocol 54)
  • Succinate solution (see recipe)
  • INT solution (see recipe)
  • Stop solution I (see recipe)
  • Spectrophotometer and glass cuvettes

Support Protocol 2: β‐Galactosidase Assay for Lysosomes

  Materials
  • Mitochondrial gradient fraction (see Basic Protocols protocol 11 to protocol 54)
  • Substrate solution (see recipe)
  • Stop solution II (see recipe)
  • Spectrophotometer and plastic cuvettes

Support Protocol 3: Catalase Assay for Peroxisomes

  Materials
  • Mitochondrial gradient fraction (see Basic Protocols protocol 11 to protocol 54)
  • Stock peroxide solution (see recipe)
  • Tris/BSA solution (see recipe)
  • Titanium oxysulfate reagent (see recipe)
  • Sample buffer (see recipe)
  • Spectrophotometer
NOTE: Carry out all operations on ice in 2‐ml microcentrifuge tubes.
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Figures

Videos

Literature Cited

Literature Cited
   Baudhuin, P. 1974. Isolation of rat liver peroxisomes. Methods Enzymol. 31:356‐368.
   Beauvoit, B., Rigoulet, M., Guerin, B., and Canioni, P. 1989. Polyphosphates, a source of high energy phosphate in yeast mitochondria: A 31P NMR study. FEBS Lett. 252:17‐21.
   Diczfalusy, U. and Alexson, S.E.H. 1988. Peroxisomal chain shortening of prostaglandin F2αJ. Lipid Res. 29:1629‐1636.
   Dobrota, M. and Hinton, R. 1992. Conditions for density gradient separations. In Preparative Centrifugation: A Practical Approach (D. Rickwood, ed.) pp. 77‐142. IRL Press, Oxford.
   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.
   Graham, J.M. 1993. The identification of subcellular fractions from mammalian cells. In Methods in Molecular Biology, Vol.19 (J.M. Graham and J.A. Higgins, eds.) pp. 1‐18. Humana Press, Totowa, N.J.
   Graham, J., Ford, T., and Rickwood, D. 1990. Isolation of the major subcellular organelles from mouse liver using Nycodenz gradients without the use of an ultracentrifuge. Anal. Biochem. 187:318‐323.
   Graham, J., Ford, T., and Rickwood, D. 1994. The preparation of subcellular organelles from mouse liver in self‐generated gradients of iodixanol. Anal. Biochem. 220:267‐373.
   Hovius, R., Lambrechts, H., Nicolay, K., and de Kruijff, B. 1990. Improved methods to isolate and subfractionate rat liver mitochondria. Lipid composition of the inner and outer membrane. Biochim. Biophys. Acta 1021:217‐226
   Leighton, F., Poole, B., Beaufay, H., Baudhuin, P., Coffey, J.W., Fowler, S., and de Duve, C. 1968. The large‐scale separation of peroxisomes, mitochondria and lysosomes from the livers of rats injected with Triton WR 1339. J. Cell Biol. 37:482‐513.
   Osmundsen, H. 1982. Factors which can influence β‐oxidation by peroxisomes isolated from livers of clofibrate‐treated rats. Some properties of peroxisomal fractions isolated in a self‐generated Percoll gradient by vertical rotor centrifugation. Int. J. Biochem. 14:905‐914.
   Patel, B., Costi, A., Hardy, L., and Mowbray, J. 1991. The characterization of a new enzyme from rat liver mitochondria, oligophosphoglyceroyl‐ATP 3′‐phosphodiesterase. Biochem. J. 274:275‐279.
   Reinhart, P.H., Taylor, W.M., and Bygrave, F.L. 1982. A procedure for the rapid preparation of mitochondria from rat liver. Biochem. J. 204:731‐735.
   Sims, N.R. 1990. Rapid isolation of metabolically active mitochondria from rat forebrain and subregions using Percoll density gradient centrifugation. J. Neurochem. 55:698‐705.
   Singh, H., Derwas, N., and Poulos, A. 1987. Very long chain fatty acid β‐oxidation by rat liver mitochondria and peroxisomes. Arch. Biochem. Biophys. 259:382‐290.
   Symons, L.J. and Jonas, A.J. 1987. Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients. Anal. Biochem. 164:382‐390.
   Vamecq, J. and Van Hoof, F. 1984. Implication of a peroxisomal enzyme in the catabolism of glutaryl‐CoA. Biochem. J. 221:203‐211.
   Wattiaux, R. and Wattiaux‐De Coninck, S. 1983. Separation of cell organelles. In Iodinated Density Gradient Media: A Practical Approach (D. Rickwood, ed.) pp. 119‐137. IRL Press, Oxford.
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