Journal of Inherited Metabolic Disease

, 34:741

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

DOI: 10.1007/s10545-011-9315-7

Cite this article as:
Smet, J., De Paepe, B., Seneca, S. et al. J Inherit Metab Dis (2011) 34: 741. doi:10.1007/s10545-011-9315-7

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.

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

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