Complex III staining in blue native polyacrylamide gels

  • Joél Smet
  • Boel De Paepe
  • Sara Seneca
  • Willy Lissens
  • Heike Kotarsky
  • Linda De Meirleir
  • Vineta Fellman
  • Rudy Van Coster
Original Article


For more than a decade now blue native polyacrylamide gel electrophoresis (BN-PAGE) has been used for the study of the oxidative phosphorylation (OXPHOS) complexes. Catalytic activities of complexes I, II, IV and V can be assessed, after separation by gel electroforesis, by incubation of the BN-PAGE gel in specific staining solutions. However, until now, a reliable staining method for testing ubiquinol cytochrome c oxidoreductase (complex III) activity by BN-PAGE gel techniques was not available. In addition, spectrophotometric methods currently in use for detection of complex III deficiency in patients are not very sensitive. Here, we describe a newly developed diagnostic method for visualization of complex III activity by direct in-gel evaluation of ubiquinol cytochrome oxidoreductase activity. We validated the method by reporting the results in six patients with previously characterised complex III defects.



Human bc1 synthesis like gene


Blue native polyacrylamide gel electrophoresis


High resolution Clear Native Electrophoresis


Mitochondrial DNA


Oxidative phosphorylation


Gene coding for polymerase gamma


Thymidine kinase gene




Ubiquinol-cytochrome c reductase binding protein


  1. Barel O, Shorer Z, Flusser H et al (2008) Mitochondrial complex III deficiency associated with a homozygous mutation in UQCRQ. Am J Hum Genet 82:1211–1216PubMedCrossRefGoogle Scholar
  2. Blakely EL, Mitchell AL, Fisher N et al (2005) A mitochondrial cytochrome b mutation causing severe respiratory chain enzyme deficiency in humans and yeast. FEBS J 272:3583–3592PubMedCrossRefGoogle Scholar
  3. Calvaruso MA, Smeitink J, Nijtmans L (2008) Electrophoresis techniques to investigate defects in oxidative phosphorylation. Methods 46:281–287PubMedCrossRefGoogle Scholar
  4. Benit P, Goncalves S, Philippe Dassa E et al (2006) Three spectrophotometric assays for the measurement of the five respiratory chain complexes in minuscule biological samples. Clin Chim Acta 374:81–86PubMedCrossRefGoogle Scholar
  5. Benit P, Lebon S, Rustin P (2009) Respiratory-chain diseases related to complex III deficiency. Biochim Biophys Acta 1793:181–185PubMedCrossRefGoogle Scholar
  6. de Lonlay P, Valnot I, Barrientos A et al (2001) A mutant mitochondrial respiratory chain assembly protein causes complex III deficiency in patients with tubulopathy, encephalopathy and liver failure. Nat Genet 29:57–60PubMedCrossRefGoogle Scholar
  7. De Meirleir L, Seneca S, Damis E et al (2003) Clinical and diagnostic characteristics of complex III deficiency due to mutations in the BCS1L gene. Am J Med Genet A 121A:126–131PubMedCrossRefGoogle Scholar
  8. Díaz F, Barrientos A, Fontanesi F (2009) Evaluation of the mitochondrial respiratory chain and oxidative phosphorylation system using blue native gel electrophoresis. Curr Protoc Hum Genet. 19:19.4Google Scholar
  9. Fellman V (2002) The GRACILE syndrome, a neonatal lethal metabolic disorder with iron overload. Blood Cells Mol Dis 29:444–450PubMedCrossRefGoogle Scholar
  10. Fellman V, Lemmelä S, Sajantila A, Pihko H, Järvelä I (2008) Screening of BCS1L mutations in severe neonatal disorders suspicious for mitochondrial cause. J Hum Genet 53:554–558PubMedCrossRefGoogle Scholar
  11. Haut S, Brivet M, Touat G et al (2003) A deletion in the human QP-C gene causes a complex III deficiency resulting in hypoglycaemia and lactic acidosis. Hum Genet 113:118–122PubMedGoogle Scholar
  12. Hinson JT, Fantin VR, Schonberger J et al (2007) Missense mutations in the BCS1L gene as a cause of the Bjornstad syndrome. N Engl J Med 356:809–819PubMedCrossRefGoogle Scholar
  13. Kotarsky H, Karikoski R, Mörgelin M et al (2010) Characterization of complex III deficiency and liver dysfunction in GRACILE syndrome caused by a BCS1L mutation. Mitochondrion 10:497–509PubMedCrossRefGoogle Scholar
  14. Luo C, Long J, Liu J (2008) An improved spectrophotometric method for a more specific and accurate assay of mitochondrial complex III activity. Clin Chim Acta 395:38–41PubMedCrossRefGoogle Scholar
  15. Meulemans A, Seneca S, Smet J et al (2007a) A new family with the mitochondrial tRNAGLU gene mutation m.14709 T > C presenting with hydrops fetalis. Eur J Paediatr Neurol 11:17–20PubMedCrossRefGoogle Scholar
  16. Meulemans A, De Paepe B, De Bleecker J et al (2007b) Two novel mitochondrial DNA mutations in muscle tissue of a patient with limb-girdle myopathy. Arch Neurol 64:1339–1343PubMedCrossRefGoogle Scholar
  17. Schaefer AM, Taylor RW, Turnbull DM, Chinnery PF (2004) The epidemiology of mitochondrial disorders–past, present and future. Biochim Biophys Acta 1659:115–120PubMedCrossRefGoogle Scholar
  18. Smet J, Devreese B, van Beeumen J, Van Coster R (2005) Nondenaturing polyacrylamide gel electrophoresis as a method for studying protein interactions: applications in the analysis of mitochondrial OXPHOS complexes. In: Celis JE (ed) Cell biology: a laboratory handbook. Academic, San Diego, pp 259–264Google Scholar
  19. Van Biervliet S, Verloo P, Vande Veldel S et al (2009) Abdominal pain and vomiting as first sign of mitochondrial disease. Acta Gastroenterol Belg 72:365–368PubMedGoogle Scholar
  20. van der Westhuizen FH, Smet J, Levanets O et al (2010) Aberrant synthesis of ATP synthase resulting from a novel deletion in mitochondrial DNA in an African patient with progressive external ophthalmoplegia. J Inherit Metab Dis 2010 Jan 16 [Epub ahead of print]Google Scholar
  21. Van Coster R, Smet J, George E et al (2001) Blue native polyacrylamide gel electrophoresis: a powerful tool in diagnosis of oxidative phosphorylation defects. Pediatr Res 50:658–665PubMedCrossRefGoogle Scholar
  22. Visapää I, Fellman V, Vesa J et al (2002) GRACILE syndrome, a lethal metabolic disorder with iron overload, is caused by a point mutation in BCS1L. Am J Hum Genet 71:863–876PubMedCrossRefGoogle Scholar
  23. Wittig I, Karas M, Schägger H (2007) High resolution clear native electrophoresis for in-gel functional assays and fluorescence studies of membrane protein complexes. Mol Cell Proteomics 6:1215–1225PubMedCrossRefGoogle Scholar
  24. Zerbetto E, Vergani L, Dabbeni-Sala F (1997) Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels. Electrophoresis 18:2059–2064PubMedCrossRefGoogle Scholar

Copyright information

© SSIEM and Springer 2011

Authors and Affiliations

  • Joél Smet
    • 1
  • Boel De Paepe
    • 1
  • Sara Seneca
    • 2
  • Willy Lissens
    • 2
  • Heike Kotarsky
    • 3
  • Linda De Meirleir
    • 2
  • Vineta Fellman
    • 3
    • 4
  • Rudy Van Coster
    • 1
  1. 1.Department of Pediatrics, Division of Pediatric Neurology and MetabolismGhent University HospitalGhentBelgium
  2. 2.Center for Medical Genetics, UZ Brussel and Reproduction and Genetics (REGE)Vrije Universiteit BrusselBrusselsBelgium
  3. 3.Department of Pediatrics, Clinical SciencesLund UniversityLundSweden
  4. 4.Department of Pediatrics, Clinical SciencesUniversity of HelsinkiHelsinkiFinland

Personalised recommendations