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Functional assays in high‐resolution clear native gels to quantify mitochondrial complexes in human biopsies and cell lines
Author(s) -
Wittig Ilka,
Carrozzo Rosalba,
Santorelli Filippo M.,
Schägger Hermann
Publication year - 2007
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.200700367
Subject(s) - mitochondrion , cell culture , oxidative phosphorylation , cell , microbiology and biotechnology , atp synthase , gel electrophoresis , biochemistry , biology , high resolution , mitochondrial dna , enzyme , respiratory chain , chemistry , gene , genetics , remote sensing , geology
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.