Zischka Hans, Lichtmannegger Josef, Jägemann Nora, Jennen Luise, Hamöller Daniela, Huber Evamaria, Walch Axel, Summer Karl H, Göttlicher Martin
GSF-National Research Center for Environment and Health, Neuherberg, Germany.
Methods Mol Biol. 2008;424:333-48. doi: 10.1007/978-1-60327-064-9_26.
This protocol describes the purification of mitochondria from rat liver with the aid of zone electrophoresis in a free flow device (ZE-FFE). Starting from liver homogenate, cell debris and nuclei are removed by low speed centrifugation. A crude mitochondrial fraction is obtained by medium speed centrifugation and is further purified by washing followed by a Nycodenz gradient centrifugation. Lysosomes and microsomes are located at the upper parts of the gradient, whereas mitochondria are found in the medium part of the gradient. A subsequent purification step with ZE-FFE efficiently removes remaining lysosomes and microsomes and, importantly, damaged mitochondrial structures. The resulting purified mitochondria can be concentrated by centrifugation and used for further experiments. Finally, possible modifications of this protocol with respect to the isolation of pure lysosomes are discussed.
本方案描述了借助自由流动装置（ZE-FFE）中的区带电泳从大鼠肝脏中纯化线粒体的方法。从肝脏匀浆开始，通过低速离心去除细胞碎片和细胞核。通过中速离心获得粗线粒体组分，并通过洗涤进一步纯化，随后进行 Nycodenz 梯度离心。溶酶体和微粒体位于梯度的上部，而线粒体则存在于梯度的中部。随后用 ZE-FFE 进行的纯化步骤可有效去除残留的溶酶体和微粒体，重要的是，还能去除受损的线粒体结构。所得纯化的线粒体可通过离心浓缩并用于进一步实验。最后，讨论了该方案在分离纯溶酶体方面可能的改进。
