Functional assays in high-resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines.
We introduce high resolution clear native electrophoresis (CNE) as a powerful technique to resolve enzymatically active mitochondrial complexes from cultured human cell lines and skeletal muscle biopsy samples. Quantitative enzymatic assays can be performed using small amounts of cultured cells with low mitochondria content, for example, around 10 mg of sedimented osteosarcoma cells (wet weight) which is equivalent to around 10 million cells. High resolution CNE offers general advantages for in-gel catalytic activity assays compared to blue native electrophoresis. It seems especially suited for assaying mitochondrial ATP synthase and respiratory chain complexes I and II in cell models of human mitochondrial disorders and for detailed analyses of patient cells and tissues with defects in oxidative phosphorylation.
Wittig
Ilka
I
Molekulare Bioenergetik, Zentrum der Biologischen Chemie, Centre of Excellence Macromolecular Complexes, Universität Frankfurt, Frankfurt, Germany. wittig@zbc.kgu.de
Carrozzo
Rosalba
R
Santorelli
Filippo M
FM
Schägger
Hermann
H
D001681:Biological Assay; D001706:Biopsy; D002384:Catalysis; D002460:Cell Line; D004072:Digitonin / Q000737:chemistry; D042967:Electron Transport Complex I / Q000032:analysis*; D042963:Electron Transport Complex II / Q000032:analysis*; D014450:Electron Transport Complex III / Q000032:analysis*; D003576:Electron Transport Complex IV / Q000032:analysis*; D015180:Electrophoresis, Gel, Two-Dimensional; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D008931:Mitochondria, Muscle / Q000201:enzymology; D024101:Mitochondrial Proteins / Q000032:analysis*; D010085:Oxidative Phosphorylation; D015203:Reproducibility of Results; D013048:Specimen Handling
D024101:Mitochondrial Proteins; D042963:Electron Transport Complex II; D003576:Electron Transport Complex IV; D042967:Electron Transport Complex I; D014450:Electron Transport Complex III; D004072:Digitonin