User Ratings

Easy to Follow Achieved Expected Results Overall Rating Add your comments

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

Francisca Díaz¹, Antoni Barrientos¹, Flavia Fontanesi¹

¹University 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

GO TO THE FULL TEXT:

PDF or HTML at Wiley Online Library

Are you the author of this protocol? Login or register and return to this page.

Learn more about the author(s):

Antoni Barrientos

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

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
MgSO₄
ATP
0.2% Pb(NO₃)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)

Looking for materials? Suppliers Guide

GO TO THE FULL PROTOCOL:

PDF or HTML at Wiley Online Library

Figures

Figure 19.4.1

Analysis of mitochondria by blue native gel electrophoresis (BNGE). (A) Mouse muscle mitochondria separated by BNGE and stained with Coomassie blue to visualize respiratory complexes: lane 1: control mouse; lane 2: COX deficient mouse; lane 3: native high-molecular-weight marker. (B) BN-western blot using Invitrogen 4-16 gradient gel and Antibody Cocktail from Mitosciences. The samples are mitochondrial fractions from mouse fibroblasts. Lanes 1 and 3: control cells. Lanes 2 and 4: fibroblasts deficient in CI and CIII. (C) In gel activity stain of respiratory complexes of mouse liver mitochondria. In CV activity stain the F1 (lower band) dissociates from the fully assembled complex (arrow) and retains ATP hydrolysis activity.

View Image

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.
	Hartwig, S., Feckler, C., Lehr, S., Wallbrecht, K., Wolgast, H., Muller-Wieland, D., and Kotzka, J. 2009. A critical comparison between two classical and a kit-based method for mitochondria isolation. Proteomics. 9:3209-3214.
	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.