Zischka Hans, Kinkl Norbert, Braun Ralf J, Ueffing Marius
GSF-National Research Center for Environment and Health, Institute of Toxicology, Munich-Neuherberg, Germany.
Methods Mol Biol. 2008;432:51-64. doi: 10.1007/978-1-59745-028-7_4.
This chapter describes the isolation of yeast mitochondria by differential centrifugation followed by mitochondrial purification through zone electrophoresis (ZE) using a free flow device (FFE). Starting from a yeast colony, cultures are grown under respiratory conditions to logarithmic phase. Cells are collected, their cell walls enzymatically disintegrated and the resulting spheroplasts are homogenized. Mitochondria are pre-fractionated from this homogenate by differential centrifugation. With the focus on further purification, pre-fractionated mitochondria are subjected to ZE-FFE. In ZE-FFE, mitochondria are transported with the buffer flow through the separation chamber and purified from contaminants by specific deflection through a perpendicularly oriented electric field. The purified mitochondria can be collected by centrifugation and used for further experiments and analysis such as sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, 2-DE or electron microscopy.
本章介绍了通过差速离心法分离酵母线粒体，随后使用自由流动装置（FFE）通过区带电泳（ZE）对线粒体进行纯化的方法。从酵母菌落开始，在呼吸条件下培养至对数期。收集细胞，酶解细胞壁，将所得原生质体匀浆。通过差速离心从该匀浆中初步分离线粒体。为了进一步纯化，将初步分离的线粒体进行ZE-FFE。在ZE-FFE中，线粒体随缓冲液流通过分离室，并通过垂直定向的电场进行特定偏转，从而从污染物中纯化出来。纯化后的线粒体可通过离心收集，并用于进一步的实验和分析，如十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）、免疫印迹、双向电泳（2-DE）或电子显微镜检查。
