Afanasyeva M A, Ustiugova A S, Golyshev S A, Kopylov A T, Bogolyubova A V, Demin D E, Belousov P V, Schwartz A M
Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, 119991, Russia.
Biochemistry (Mosc). 2018 Jan;83(1):76-85. doi: 10.1134/S0006297918010108.
Ultracentrifugation on a density gradient remains the only reliable way to obtain highly pure mitochondria preparations. However, it is not readily available for any laboratory and has a serious disadvantage of providing low mitochondria yield, which can be critical when working with limited starting material. Here we describe a combined method for isolation of mitochondria for proteomic studies that includes cell disruption by sonication, differential centrifugation, and magnetic separation. Our method provides remarkable enrichment of mitochondrial proteins as compared to differential centrifugation, magnetic separation, or their combination, and it enables the strongest depletion of cytoplasmic components, as assessed by two-dimensional electrophoresis, mass spectrometry, and Western blot. It also doubles the yield of mitochondria. However, our method should not be used for functional studies as most of the isolated organelles demonstrate disturbed structure in electron microphotographs.
在密度梯度上进行超速离心仍然是获得高纯度线粒体制剂的唯一可靠方法。然而，并非任何实验室都能轻易进行，且存在严重缺点，即线粒体产量低，在使用有限起始材料时这可能至关重要。在此，我们描述一种用于蛋白质组学研究的线粒体分离组合方法，该方法包括通过超声处理进行细胞破碎、差速离心和磁分离。与差速离心、磁分离或它们的组合相比，我们的方法能显著富集线粒体蛋白质，并且通过二维电泳、质谱和蛋白质印迹评估，它能最有效地去除细胞质成分。它还使线粒体产量增加一倍。然而，我们的方法不应用于功能研究，因为在电子显微镜照片中，大多数分离出的细胞器显示出结构紊乱。
