ANALYSIS OF OXIDATIVE-PHOSPHORYLATION COMPLEXES IN CULTURED HUMAN FIBROBLASTS AND AMNIOCYTES BY BLUE-NATIVE-ELECTROPHORESIS USING MITOPLASTS ISOLATED WITH THE HELP OF DIGITONIN
ANALYSIS OF OXIDATIVE-PHOSPHORYLATION COMPLEXES IN CULTURED HUMAN FIBROBLASTS AND AMNIOCYTES BY BLUE-NATIVE-ELECTROPHORESIS USING MITOPLASTS ISOLATED WITH THE HELP OF DIGITONIN
复制标题
DOI：
10.1006/abio.1995.1523
复制
发表时间：
1995-10-10
影响因子：
2.9
通讯作者：
HOUSTEK, J
中科院分区：
文献类型：
--
作者：
KLEMENT, P;NIJTMANS, LGJ;HOUSTEK, J
Schagger和von Jagow的电泳方法（Anal. Biochem.199，223-231（1991）），以允许分析培养的人皮肤成纤维细胞和成纤维细胞中的呼吸链酶和ATP合酶。用毛地黄皂苷分级细胞，分离有丝分裂体并用于电泳，当0.8-1.6 mg毛地黄皂苷/完整的复合物I、III、IV和V在第一维通过蓝色天然聚丙烯酰胺凝胶电泳（PAGE）清楚地分离，在第二维通过三羟甲基甘氨酸-十二烷基硫酸钠-PAGE清楚地分离它们的单个亚基。大约10（6）个成纤维细胞或巨噬细胞（0.4-0.6 mg蛋白质）足以使用通过染色和通过蛋白质印迹的检测来完全分析氧化磷酸化复合物。用其它细胞类型获得相当的分辨率。细胞色素c氧化酶缺乏症患者的成纤维细胞的研究证明了该方法用于诊断线粒体疾病的有用性。(C)出版社：Academic Press
The electrophoretic method of Schagger and von Jagow (Anal. Biochem. 199, 223-231 (1991)) was adapted to allow analysis of enzymes of the respiratory chain and the ATP-synthase in cultured human skin fibroblasts and amniocytes, The cells were fractionated with digitonin and mitoplasts were isolated and used for electrophoresis, The purification of mitoplasts and the resolution by electrophoresis of the oxidative phosphorylation complexes were optimal when 0.8-1.6 mg of digitonin/mg protein was used, Intact complexes I, III, IV, and V were clearly separated by blue native-polyacrylamide gel electrophoresis (PAGE) in the first dimension and their individual subunits by tricine-sodium dodecyl sulfate-PAGE in the second dimension. Approximately 10(6) fibroblasts or amniocytes (0.4-0.6 mg protein) were sufficient for complete analysis of the oxidative phosphorylation complexes using detection by staining and by Western blotting, Comparable resolution was obtained with other cell types. Studies of fibroblasts from patients with cytochrome c oxidase deficiency demonstrated the usefulness of the method for diagnosis of mitochondrial disorders. (C) 1995 Academic Press, Inc.