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Mitochondrial Purification by Two Times Sucrose Mitochondrial Purification by Two Times Sucrose
Zhou Wangzhan, Huang Jun, Kuang Lingyun, Zhou Xiaojing, Guo Nengneng, Lü Jianxin*, Liu Danhui*
Institute of Molecular & Cellular Medicine, Zhejiang Provincial Key Laboratory of Medical Genetics,Wenzhou Medical College, Wenzhou 325035, China
Abstract: Mitochondrial fraction produced by differential centrifugation is often contaminated by other organelles and cytosolic components， and its further applications are thus limited. Density-gradient centrifugation is the most used strategy to improve the mitochondrial purity. However， mitochondria fraction obtained by density-gradient centrifugation is still slightly contaminated. In this report， we found an additional discontinuous sucrose density-gradient centrifugation will significantly improve the mitochondrial purity. Transmission electron microscopy and Western blot results suggested mitochondrial fraction isolated by differential centrifugation contained large amount of other organelles and cytosolic components. The isolated mitochondria were still contaminated by other impurities after one time discontinuous sucrose density-gradient centrifugation， although its purity was significantly enhanced. Impressively， the mitochondrial purity was highly improved by the second time centrifugation， and the yield was only slightly affected. The results of mitochondrial membrane potential and succinate dehydrogenase activity assay indicated that the mitochondrial membrane integrity and function were kept well with prolonged experimental procedure. Thus we suggest one or two additional density-gradient centrifugations could easily improve the mitochondria purity to facilitate further assays.
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