Isolation of Mitochondria from Tissues and Cells by Differential Centrifugation

John M. Graham1

1 Liverpool John Moores University, Liverpool
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.3
DOI:  10.1002/0471143030.cb0303s04
Online Posting Date:  May, 2001
GO TO THE FULL TEXT: PDF or HTML at Wiley Online Library

Abstract

Differential centrifugation is used to prepare a “heavy” mitochondrial fraction from liver. These mitochondria are relatively pure, highly coupled, and suitable for respiratory studies. This unit presents protocols for isolation of beef heart mitochondria (also suitable for respiratory studies), skeletal muscle mitochondria, and mitochondria from cultured cells can also be isolated from homogenates by differential centrifugation. Differential centrifugation, which separates cellular organelles based on sedimentation velocity, is a rapid method for preparing mitochondria for metabolic studies.

     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Table of Contents

  • Basic Protocol 1: Preparation of the Heavy Mitochondrial Fraction from Rat Liver
  • Basic Protocol 2: Large‐Scale Preparation of Mitochondria from Bovine Heart
  • Basic Protocol 3: Preparation of Mitochondria from Skeletal Muscle
  • Basic Protocol 4: Preparation of Mitochondria from Cultured Cells
  • Basic Protocol 5: Preparation of Mitochondria from Yeast (Saccharomyces cerevisiae)
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Materials

Basic Protocol 1: Preparation of the Heavy Mitochondrial Fraction from Rat Liver

  Materials
  • 150‐ to 200‐g male Sprague‐Dawley rat
  • Liver homogenization medium (LHM; see recipe), ice cold
  • Dissecting tools
  • Potter‐Elvehjem homogenizer (∼0.09‐mm clearance; 25‐ml working volume)
  • Overhead high‐torque electric motor (thyristor‐controlled)
  • Low‐speed centrifuge with swinging bucket rotor and appropriate tubes
  • High‐speed centrifuge with fixed‐angle rotor and 40‐ to 50‐ml polycarbonate tubes
  • Vacuum pump
  • Dounce homogenizer (30‐ to 40‐ml volume) with loose‐fitting pestle (Wheaton type B)

Basic Protocol 2: Large‐Scale Preparation of Mitochondria from Bovine Heart

  Materials
  • Bovine heart, freshly isolated
  • Heart wash buffer (see recipe)
  • 2.0 M Tris base
  • Sucrose/succinate solution (SS; see recipe)
  • Commercial mincer with a total capacity of 2 to 3 liters
  • Waring blender or other large‐capacity rotating blades homogenizer
  • Cotton muslin
  • Low‐speed centrifuge with swinging‐bucket rotor and 250‐ to 750‐ml bottles
  • High‐speed centrifuge with fixed‐angle rotor
  • Glass rod
  • Dounce homogenizer (50‐ml volume) with loose‐fitting pestle (Wheaton type B)

Basic Protocol 3: Preparation of Mitochondria from Skeletal Muscle

  Materials
  • 150‐ to 200‐g male Sprague‐Dawley rat
  • Muscle wash buffer (see recipe)
  • Muscle homogenization medium I (see recipe)
  • Muscle homogenization medium II (see recipe)
  • Dissecting tools
  • Potter‐Elvehjem homogenizer (∼0.3‐mm clearance, 40‐ml working volume)
  • Overhead high‐torque electric motor (thyristor‐controlled)
  • Fine nylon mesh (200‐µm pore size)
  • Low‐speed centrifuge with swinging‐bucket rotor and appropriate tubes
  • High‐speed centrifuge with fixed‐angle rotor
  • Dounce homogenizer (30‐ml volume) with loose‐fitting pestle (Wheaton type B)

Basic Protocol 4: Preparation of Mitochondria from Cultured Cells

  Materials
  • Confluent monolayer cultured cells (total 2 × 108)
  • PBS ( appendix 2A)
  • Cell homogenization medium (CHM; see reciperecipe)
  • recipeCHM containing 1 M sucrose
  • Sucrose/Mg2+ medium (see recipe)
  • Mitochondrial suspension medium I (see recipe)
  • Rubber policeman
  • Potter‐Elvehjem homogenizer (∼0.09‐mm clearance, 10‐ to 15‐ml working volume)
  • Overhead high‐torque electric motor (thyristor‐controlled)
  • Low‐speed centrifuge with swinging‐bucket rotor and appropriate tubes
  • High‐speed centrifuge with fixed‐angle rotor and appropriate tubes
  • Dounce homogenizer (5‐ to 10‐ml volume) with loose‐fitting pestle (Wheaton type B)

Basic Protocol 5: Preparation of Mitochondria from Yeast (Saccharomyces cerevisiae)

  Materials
  • Yeast cultures in early‐ to mid‐log phase, grown in YPD medium (1% yeast extract/2% Bacto Peptone/2% glucose; see unit 1.6)
  • DTT buffer (see recipe)
  • Yeast sorbitol buffer (see recipe)
  • Zymolase‐100T (from Arthrobacter luteus, 100,000 U/g; e.g., ICN Biomedicals, Sigma)
  • Spheroplast homogenization medium (see recipe), ice cold
  • Mitochondrial suspension medium II (see recipe), ice cold
  • Low‐speed centrifuge with swinging‐bucket rotor and appropriate tubes
  • High‐speed centrifuge with fixed‐angle rotor and appropriate tubes
  • 30°C incubator
  • Dounce homogenizer (5‐ to 10‐ml volume) with loose‐fitting pestle (Wheaton type B)
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library

Figures

Videos

Literature Cited

Literature Cited
   Attardi, G. and Ching, E. 1979. Biogenesis of mitochondrial protein in HeLa cells. Methods Enzymol. 56:66‐79.
   Bhattacharya, S.K., Thakar, J.H., Johnson, P.L., and Shanklin, D.R. 1991. Isolation of skeletal muscle mitochondria from hamsters using an ionic medium containing EDTA and Nagarse. Anal. Biochem. 192:344‐349.
   Evans, W.H. 1992. Isolation and characterization of membranes and cell organelles. In Preparative Centrifugation: A Practical Approach (D. Rickwood, ed) pp. 233‐270. IRL Press, Oxford.
   Goldberg, D.E. and Kornfeld, S. 1983. Evidence for extensive subcellular organization of asparagine‐linked oligosaccharide processing and lysosomal enzyme phosphorylation. J. Biol. Chem. 258:3159‐3165.
   Goud, B., Salminen, A., Walworth, N.C., and Novick, P.J. 1988. A GTP‐binding protein required for secretion rapidly associates with secretory vesicles and the plasma membrane in yeast. Cell 53:753‐768.
   Graham, J.M. 1997. Homogenization of tissues and cells. In Subcellular Fractionation: A Practical Approach (J.M. Graham and D. Rickwood, eds.) pp. 1‐29. IRL Press, Oxford.
   Hinton, R.H. and Mullock, B.M. 1997. Isolation of subcellular fractions. In Subcellular Fractionation: A Practical Approach. (J.M. Graham and D. Rickwood, eds.) pp. 31‐69. IRL Press, Oxford.
   Marsh, M., Schmid, S., Kern, H., Harms, E., Male, P., Mellman, I., and Helenius, A. 1987. Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresis. J. Cell Biol. 104:875‐886.
   Rice, J.E. and Lindsay, J.G. 1997. Subcellular fractionation of mitochondria. In Subcellular Fractionation: A Practical Approach (J.M. Graham and D. Rickwood, eds.) pp. 107‐142. IRL Press, Oxford.
   Smith, A.L. 1967. Preparation and properties and conditions for assay of mitochondria: Slaughterhouse material small scale. Methods Enzymol. 10:81‐86.
GO TO THE FULL PROTOCOL:
PDF or HTML at Wiley Online Library