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Complex III staining in blue native polyacrylamide gels
Author(s) -
Smet Joél,
De Paepe Boel,
Seneca Sara,
Lissens Willy,
Kotarsky Heike,
De Meirleir Linda,
Fellman Vineta,
Van Coster Rudy
Publication year - 2011
Publication title -
journal of inherited metabolic disease
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.462
H-Index - 102
eISSN - 1573-2665
pISSN - 0141-8955
DOI - 10.1007/s10545-011-9315-7
Subject(s) - ubiquinol , polyacrylamide gel electrophoresis , staining , oxidoreductase , gel electrophoresis , oxidative phosphorylation , silver stain , cytochrome , cytochrome c , chemistry , biochemistry , coenzyme q – cytochrome c reductase , microbiology and biotechnology , biology , enzyme , mitochondrion , genetics
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.