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Complex III staining in blue native polyacrylamide gels

  • Original Article
  • Published:
Journal of Inherited Metabolic Disease

Abstract

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.

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Fig. 1

Abbreviations

BCS1L :

Human bc1 synthesis like gene

BN-PAGE:

Blue native polyacrylamide gel electrophoresis

hrCNE:

High resolution Clear Native Electrophoresis

mtDNA:

Mitochondrial DNA

OXPHOS:

Oxidative phosphorylation

POLG :

Gene coding for polymerase gamma

TK2 :

Thymidine kinase gene

TMB:

3,3′,5,5′-tetramethylbenzidine

UQCRB:

Ubiquinol-cytochrome c reductase binding protein

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Acknowledgments

We thank Mr Christophe Meul, professor in Integrated Digital Techniques and Fine Arts, for his help in Photoshop® for digital processing of images. We also thank Mary Phelan for her help with correcting the English language. This study was supported by a grant from the Fund for Scientific Research Belgium (FWO), contract grant number G.0666.06 (Universiteit Gent and Vrije Universiteit Brussel) and from ‘KidAuQuai’, Ghent, Belgium, and the Medical Society of Finland (Finska Läkaresällskapet).

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Authors and Affiliations

  1. Department of Pediatrics, Division of Pediatric Neurology and Metabolism, Ghent University Hospital, De Pintelaan 185, 9000, Ghent, Belgium

    Joél Smet, Boel De Paepe & Rudy Van Coster

  2. Center for Medical Genetics, UZ Brussel and Reproduction and Genetics (REGE), Vrije Universiteit Brussel, Brussels, Belgium

    Sara Seneca, Willy Lissens & Linda De Meirleir

  3. Department of Pediatrics, Clinical Sciences, Lund University, Lund, Sweden

    Heike Kotarsky & Vineta Fellman

  4. Department of Pediatrics, Clinical Sciences, University of Helsinki, Helsinki, Finland

    Vineta Fellman

Authors
  1. Joél Smet
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  2. Boel De Paepe
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  3. Sara Seneca
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  4. Willy Lissens
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  5. Heike Kotarsky
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  6. Linda De Meirleir
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  7. Vineta Fellman
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  8. Rudy Van Coster
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Corresponding author

Correspondence to Rudy Van Coster.

Additional information

Communicated by: John Christodoulou.

References to electronic databases: Mitochondrial complex III deficiency: OMIM 124000. UQCRQ encoding subunit 7: OMIM 61280. UQCRB encoding ubiquinol-cytochrome c reductase binding protein: OMIM 191330. Cytochrome b of Complex IIII, MTCYB: OMIM 516020. GRACILE syndrome (Growth retardation, Amino aciduria, Cholestasis, Iron overload, Lactic acidosis): OMIM 603358. Bjornstad syndrome: OMIM 262000. NADH:ubiquinone oxidoreductase (complex I): EC 1.6.5.3. Succinate:ubiquinone oxidoreductase (complex II): EC 1.3.5.1. Ubiquinol:ferricytochrome-c oxidoreductase (complex III): EC 1.10.2.2. Ferrocytochrome-c:oxygen oxidoreductase (complex IV): EC 1.9.3.1. ATP phosphohydrolase (complex V): EC 3.6.1.3. Deoxynucleoside-triphosphate:DNA deoxynucleotidyltransferase (DNA-directed; DNA polymerase gamma): EC 2.7.7.7. Mitochondrial thymidine kinase 2: EC 2.7.1.21.

Competing of interest: None declared.

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Smet, J., De Paepe, B., Seneca, S. et al. Complex III staining in blue native polyacrylamide gels. J Inherit Metab Dis 34, 741–747 (2011). https://doi.org/10.1007/s10545-011-9315-7

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  • DOI: https://doi.org/10.1007/s10545-011-9315-7

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