Lee Ken-Wing, Bogenhagen Daniel F
Department of Pharmacological Sciences, Stony Brook University, Stony Brook, NY, 11794-8651, USA.
Memorial Sloan Kettering Cancer Institute, New York, NY, USA.
Methods Mol Biol. 2016;1351:67-79. doi: 10.1007/978-1-4939-3040-1_6.
Isolation of mitochondria from cultured cells and animal tissues for analysis of nucleic acids and bona fide mitochondrial nucleic acid binding proteins and enzymes is complicated by contamination with cellular nucleic acids and their adherent proteins. Protocols presented here allow for quick isolation of mitochondria from a small number of cells and for preparation of highly purified mitochondria from a larger number of cells using nuclease treatment and high salt washing of mitochondria to reduce contamination. We further describe a method for the isolation of mitochondrial DNA-protein complexes known as nucleoids from these highly purified mitochondria using a combination of glycerol gradient sedimentation followed by isopycnic centrifugation in a non-ionic iodixanol gradient.
从培养细胞和动物组织中分离线粒体以分析核酸、真正的线粒体核酸结合蛋白和酶，会因细胞核酸及其附着蛋白的污染而变得复杂。本文介绍的方案允许从少量细胞中快速分离线粒体，并通过核酸酶处理和线粒体的高盐洗涤以减少污染，从大量细胞中制备高度纯化的线粒体。我们还描述了一种从这些高度纯化的线粒体中分离称为类核的线粒体DNA-蛋白质复合物的方法，该方法结合了甘油梯度沉降，然后在非离子型碘克沙醇梯度中进行等密度离心。
