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Quantification of muscle mitochondrial oxidative phosphorylation enzymes via histochemical staining of blue native polyacrylamide gels
Author(s) -
Zerbetto Elisabetta,
Vergani Lodovica,
DabbeniSala Federica
Publication year - 1997
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150181131
Subject(s) - oxidative phosphorylation , polyacrylamide gel electrophoresis , enzyme , polyacrylamide , staining , biochemistry , mitochondrion , skeletal muscle , biology , phosphorylation , chemistry , microbiology and biotechnology , anatomy , genetics
Blue native‐polyacrylamide gel electrophoresis is a powerful technique that enables the separation of intact multi‐subunit complexes. However, positive identification of particular enzymes generally requires further separation in a second dimension on a denaturing polyacrylamide gel. Histochemical staining is widely used to demonstrate enzyme activities in tissues, including oxidative phosphorylation enzymes. In this report, we demonstrate that the two techniques can be combined to quantify in situ mitochondrial enzymes, separated on nondenaturing polyacrylamide gels. The method gives quantitative results with human skeletal muscle as well as heart that contains higher mitochondrial numbers. Comparison of muscle from patients with oxidative phosphorylation enzyme deficiencies, such as those of two riboflavin‐responsive patients, before and after vitamin treatment, gives results in agreement with those obtained by analyzing the activity of the mitochondrial enzymes in muscle homogenates.