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Updated: Jan 23, 2026
Hybrid Clear/Blue Native Electrophoresis for the Separation and Analysis of Mitochondrial Respiratory Chain Supercomplexes
Published on: May 19, 2019
Hybrid gel electrophoresis using skin fibroblasts to aid in diagnosing mitochondrial disease
Christopher Newell1, Aneal Khan1, David Sinasac1
1Department of Medical Genetics (C.N., A.K., D.S.) and Department of Pediatrics (A.K.), Alberta Children's Hospital Research Institute, Cumming School of Medicine, University of Calgary, Canada; Atlanta (J. Shoffner), GA; Departments of Pediatrics (M.W.F., J.L.K.V.H.), Section of Clinical Genetics and Metabolism, University of Colorado; Department of Medical Genetics (S.H.), University of Alberta, Canada; Faculty of Kinesiology (J. Shearer), University of Calgary, Alberta, Canada; and Departments of Laboratory Medicine and Pathology (I.S.), University of Alberta, Edmonton, Canada.
Objective:
We developed a novel, hybrid method combining both blue-native (BN-PAGE) and clear-native (CN-PAGE) polyacrylamide gel electrophoresis, termed BCN-PAGE, to perform in-gel activity stains on the mitochondrial electron transport chain (ETC) complexes in skin fibroblasts.
Methods:
Four patients aged 46-65 years were seen in the Metabolic Clinic at Alberta Children's Hospital and investigated for mitochondrial disease and had BN-PAGE or CN-PAGE on skeletal muscle that showed incomplete assembly of complex V (CV) in each patient. Long-range PCR performed on muscle-extracted DNA identified 4 unique mitochondrial DNA (mtDNA) deletions spanning the ATP6 gene of CV. We developed a BCN-PAGE method in skin fibroblasts taken from the patients at the same time and compared the findings with those in skeletal muscle.
Results:
In all 4 cases, BCN-PAGE in skin fibroblasts confirmed the abnormal CV activity found from muscle biopsy, suggesting that the mtDNA deletions involving ATP6 were most likely germline mutations that are associated with a clinical phenotype of mitochondrial disease.
Conclusions:
The BCN-PAGE method in skin fibroblasts has a potential to be a less-invasive tool compared with muscle biopsy to screen patients for abnormalities in CV and other mitochondrial ETC complexes.
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