Analysis of Mitochondrial Respiratory Chain Supercomplexes Using Blue Native Polyacrylamide Gel Electrophoresis (BN‐PAGE)

Pooja Jha1, Xu Wang1, Johan Auwerx1

1 Laboratory for Integrative and Systems Physiology, École Polytechnique Fédérale de Lausanne, Lausanne
Publication Name:  Current Protocols in Mouse Biology
Unit Number:   
DOI:  10.1002/9780470942390.mo150182
Online Posting Date:  March, 2016
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Mitochondria are cellular organelles that harvest energy in the form of ATP through a process termed oxidative phosphorylation (OXPHOS), which occurs via the protein complexes of the electron transport chain (ETC). In recent years it has become unequivocally clear that mitochondrial complexes of the ETC are not static entities in the inner mitochondrial membrane. These complexes are dynamic and in mammals they aggregate in different stoichiometric combinations to form supercomplexes (SCs) or respirasomes. It has been proposed that the net respiration is more efficient via SCs than via isolated complexes. However, it still needs to be determined whether the activity of a particular SC is associated with a disease etiology. Here we describe a simplified method to visualize and assess in‐gel activity of SCs and the individual complexes with good resolution using blue native polyacrylamide gel electrophoresis (BN‐PAGE). © 2016 by John Wiley & Sons, Inc.

Keywords: supercomplex; in‐gel activity; mitochondria; oxidative phosphorylation; Cox7a2l; SCAFI

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Table of Contents

  • Introduction
  • Basic Protocol 1: Analysis of Respiratory Chain Supercomplexes
  • Alternate Protocol 1: CN‐Page and In‐Gel Activity of Complexes I, IV, IV + I, II, V, and III
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: Analysis of Respiratory Chain Supercomplexes

  • Mouse tissue, ∼30 mg liver; ∼15 mg from organs with high mitochondrial content, e.g., brain and heart (e.g., The Jackson Laboratory, Charles River, Taconic)
  • Isolation buffer (IB; see recipe)
  • Bovine serum albumin (BSA; Sigma‐Aldrich, cat. no. A7030)
  • Bradford protein assay (Bio‐Rad, cat. no. 500‐0006)
  • 5% digitonin (Thermo Fisher Scientific, cat. no. BN2006)
  • 4× NativePAGE sample buffer (Thermo Fisher Scientific, cat. no. BN20032)
  • NativePAGE 5% G‐250 sample additive (Thermo Fisher Scientific, cat. no. BN2004)
  • 20× NativePAGE running buffer (Thermo Fisher Scientific, cat. no. BN2001)
  • Coomassie Brilliant Blue G‐250 (SERVA, cat. no. 17524)
  • NativePAGE Novex 3% to 12% Bis‐Tris protein gels (1.0 mm, 15 well, Thermo Fisher Scientific, cat. no. BN2012BX10 or 1.0 mm, 10 well, Thermo Fisher Scientific, cat. no. BN2011BX10)
  • NativeMark Unstained Protein Standard (Thermo Fisher Scientific, cat. no. LC0725)
  • Colloidal blue staining kit (Thermo Fisher Scientific, cat. no. LC6025)
  • 20× NuPAGE transfer buffer (Thermo Fisher Scientific, cat. no. NP0006‐1)
  • Methanol (Fisher Scientific, cat. no. M/4000/17)
  • 8% acetic acid
  • WesternBreeze chromogenic kit, anti‐mouse (Thermo Fisher Scientific, cat. no. WB7103; use only for mouse; for rabbit use Thermo Fisher Scientific, cat. no. WB7105 and for goat Thermo Fisher Scientific, cat. no. WB7107, where only the secondary antibody is different; all kits contain blocker/diluent part A, blocker/diluent part B, 16× antibody wash solution, secondary antibody solution, chromogenic substrate)
  • NativePAGE anode buffer (see recipe)
  • Dark blue cathode buffer (see recipe)
  • Light blue cathode buffer (see recipe)
  • Blocking solution (see recipe)
  • OxPhos complex kit antibody cocktail (Thermo Fisher Scientific, cat. no. 45‐7999)
  • Anti‐MTCO1 antibody (Abcam, cat. no. ab14705)
  • Primary antibody solution (contains OxPhos antibody cocktail and anti‐MTCO1 antibody; see recipe)
  • Antibody wash solution (see recipe)
  • Substrates for complex I, II, IV, and V activity (see reciperecipes)
  • XCell SureLock Mini‐Cell (Thermo Fisher Scientific, cat. no. EI0001)
  • Prot/Elec tips (Bio‐Rad, cat. no. 223‐9915)
  • iBlot gel transfer device (Invitrogen, cat. no. IB1001EU)
  • iBlot gel transfer stacks, PVDF, mini (Thermo Fisher Scientific, cat. no. IB4010‐02)
  • POLYMIX PX‐SR 50 E stirrer
  • Wheaton Glass 2‐ml Potter‐Elvehjem tissue grinder set (Wheaton, cat. no. 358029)
  • 15‐ml polypropylene Falcon tubes
  • 20P pipet tips
  • Centrifuge
  • Additional reagents and equipment for anesthesia (Adams and Pacharinsak, ) and Bradford protein assay (Simonian and Smith, )
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Literature Cited

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