Evaluation of the Mitochondrial Respiratory Chain and Oxidative Phosphorylation System Using Blue Native Gel Electrophoresis

Francisca Díaz1, Antoni Barrientos1, Flavia Fontanesi1

1 University of Miami Miller School of Medicine, Miami, Florida
Publication Name:  Current Protocols in Human Genetics
Unit Number:  Unit 19.4
DOI:  10.1002/0471142905.hg1904s63
Online Posting Date:  October, 2009
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Abstract

The oxidative phosphorylation (OXPHOS) system consists of five multimeric complexes embedded in the mitochondrial inner membrane. They work in concert to drive the aerobic synthesis of ATP. Mitochondrial and nuclear DNA mutations affecting the accumulation and function of these enzymes are the most common cause of mitochondrial diseases and have also been associated with neurodegeneration and aging. For this reason, several approaches for the assessment of the OXPHOS system enzymes have been progressively developed. Based on methods described elsewhere, the use of blue native gel electrophoresis (BNGE) techniques to routinely assess the OXPHOS system and screen for enzymatic defects in homogenates or mitochondrial preparations from tissues or cultured cells is described here. Curr. Protoc. Hum. Genet. 63:19.4.1‐19.4.12. © 2009 by John Wiley & Sons, Inc.

Keywords: blue native gel electrophoresis (BNGE); electron transport chain; mitochondria; OXPHOS

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Sample Preparation
  • Basic Protocol 2: Electrophoresis and Analysis of Samples
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Sample Preparation

  Materials
  • Mitochondrial fraction, tissue culture cells, or tissue homogenates
  • Culture medium
  • Phosphate buffer saline (PBS; see appendix 2D)
  • Protease inhibitors cocktail (Roche)
  • Digitonin (see recipe)
  • Aminocaproic buffer (see recipe)
  • Bis‐Tris pH 7.0 buffer (Sigma)
  • 10% lauryl maltoside (see recipe)
  • DC protein assay kit (BioRad)
  • Serva Blue G or Coomassie Brilliant Blue G (Sigma, cat. no. B‐5133)
  • 75‐cm2 tissue culture flasks
  • 1.5‐ml microcentrifuge tubes
  • Polyvinylidene fluoride (PVDF) membrane
  • Tissue homogenizer
  • Additional reagents and equipment for cell tissue culture, counting, and trypsinization ( appendix 3G)

Basic Protocol 2: Electrophoresis and Analysis of Samples

  Materials
  • Acrylamide/bis‐acrylamide solution (see recipe)
  • 3× gel buffer (see recipe)
  • Glycerol
  • Ammonium persulfate (APS)
  • TEMED
  • 5% Serva G (see recipe)
  • Blue cathode buffer (see recipe)
  • Anode buffer (see recipe)
  • High‐molecular‐weight native markers (GE Health Care)
  • Staining solution (see recipe)
  • Destaining solution (see recipe)
  • Methanol
  • Transfer buffer (see recipe)
  • 5% milk in PBS‐Tween
  • Primary and secondary antibodies conjugated to horseradish peroxidase
  • Chemiluminescent detection kit (e.g., Supersignal from Pierce/Thermo Scientific)
  • 0.1 M Tris⋅Cl, pH 7.4 ( appendix 2D), containing 1 mg/ml nitro blue tetrazolium and 0.14 mM NADH
  • 50 mM phosphate buffer pH 7.4, 84 mM succinic acid, 0.2 mM phenazine methosulfate, 2 mg/ml nitro blue tetrazolium, 4.5 mM EDTA, and 10 mM KCN
  • 5 mg diaminobenzidine tetrahydrochloride (DAB) dissolved in 9 ml of 50 mM phosphate buffer, pH 7.4, containing 10 mg cytochrome c and 750 mg sucrose
  • Tris
  • Glycine
  • MgSO 4
  • ATP
  • 0.2% Pb(NO 3) 2
  • Mini gel system (e.g., BIORAD) with 1.5‐mm spacers
  • Gradient maker (BIORAD or Hoefer)
  • Magnetic stir bars
  • Stir plate
  • Peristaltic pump
  • Electrophoresis gel apparatus and power supply
  • Gel staining tray
  • Platform shaker
  • Kimwipes or small sponge
  • Gel photography equipment
  • Filter paper
  • PVDF membranes
  • 37°C incubator
  • Additional reagents and equipment for gel/membrane transfer (see unit 9.11)
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Figures

Videos

Literature Cited

Literature Cited
   Acin‐Perez, R., Fernandez‐Silva, P., Peleato, M.L., Perez‐Martos, A., and Enriquez, J.A. 2008. Respiratory active mitochondrial supercomplexes. Mol. Cell. 32:529‐539.
   Birch‐Machin, M.A. and Turnbull, D.M. 2001. Assaying mitochondrial respiratory complex activity in mitochondria isolated from human cells and tissues. Methods Cell Biol. 65:97‐117.
   Calvaruso, M.A., Smeitink, J., and Nijtmans, L. 2008. Electrophoresis techniques to investigate defects in oxidative phosphorylation. Methods 46:281‐287.
   Diaz, F., Fukui, H., Garcia, S., and Moraes, C.T. 2006. Cytochrome c oxidase is required for the assembly/stability of respiratory complex I in mouse fibroblasts. Mol. Cell. Biol. 26:4872‐4881.
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   Laemmli, U.K. 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature 227:680‐685.
   Nijtmans, L.G., Henderson, N.S., and Holt, I.J. 2002. Blue native electrophoresis to study mitochondrial and other protein complexes. Methods 26:327‐334.
   Rustin, P., Chretien, D., Bourgeron, T., Gerard, B., Rotig, A., Saudubray, J.M., and Munnich, A. 1994. Biochemical and molecular investigations in respiratory chain deficiencies. Clin. Chim. Acta 228:35‐51.
   Schagger, H. and Pfeiffer, K. 2000. Supercomplexes in the respiratory chains of yeast and mammalian mitochondria. EMBO J. 19:1777‐1783.
   Schagger, H. and von Jagow, G. 1991. Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form. Anal. Biochem. 199:223‐231.
   Van Coster, R., Smet, J., George, E., De Meirleir, L., Seneca, S., Van Hove, J., Sebire, G., Verhelst, H., De Bleecker, J., Van Vlem, B., Verloo, P., and Leroy, J. 2001. Blue native polyacrylamide gel electrophoresis: A powerful tool in diagnosis of oxidative phosphorylation defects. Pediatr. Res. 50:658‐665.
   Wittig, I. and Schagger, H. 2008. Features and applications of blue‐native and clear‐native electrophoresis. Proteomics 8:3974‐3990.
   Wumaier, Z., Nubel, E., Wittig, I., and Schagger, H. 2009. Chapter 8 two‐dimensional native electrophoresis for fluorescent and functional assays of mitochondrial complexes. Methods Enzymol. 456:153‐168.
   Yan, L.J. and Forster, M.J. 2009. Resolving mitochondrial protein complexes using nongradient blue native polyacrylamide gel electrophoresis. Anal. Biochem. 389:143‐149.
   Zerbetto, E., Vergani, L., and Dabbeni‐Sala, F. 1997. Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels. Electrophoresis. 18:2059‐2064.
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