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. 2020:155:3-31.
doi: 10.1016/bs.mcb.2019.10.002. Epub 2019 Dec 10.

Isolation of mitochondria from cells and tissues

Pin-Chao Liao  1 , Christian Bergamini  2 , Romana Fato  2 , Liza A Pon  3 , Francesco Pallotti  4
Affiliations

Isolation of mitochondria from cells and tissues

Pin-Chao Liao et al. Methods Cell Biol. 2020.

Abstract

Isolated mitochondria are useful to study fundamental processes including mitochondrial respiration, metabolic activity, protein import, membrane fusion, protein complex assembly, as well as interactions of mitochondria with the cytoskeleton, nuclear encoded mRNAs, and other organelles. In addition, studies of the mitochondrial proteome, phosphoproteome, and lipidome are dependent on preparation of highly purified mitochondria (Boldogh, Vojtov, Karmon, & Pon, 1998; Cui, Conte, Fox, Zara, & Winge, 2014; Marc et al., 2002; Meeusen, McCaffery, & Nunnari, 2004; Reinders et al., 2007; Schneiter et al., 1999; Stuart & Koehler, 2007). Most methods to isolate mitochondria rely on differential centrifugation, a two-step centrifugation carried out at low speed to remove intact cells, cell and tissue debris, and nuclei from whole cell extracts followed by high speed centrifugation to concentrate mitochondria and separate them from other organelles. However, methods to disrupt cells and tissue vary. Moreover, density gradient centrifugation or affinity purification of the organelle are used to further purify mitochondria or to separate different populations of the organelle. Here, we describe protocols to isolate mitochondria from different cells and tissues as well as approaches to assess the purity and integrity of isolated organelles.

Keywords: Affinity purification; Mitochondria; Subcellular fractionation; Yeast.

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Figures

Figure 1.
Figure 1.. Isolation of mitochondria using Ni-NTA magnetic beads.
(A) Scheme of mitochondrial isolation using magnetic beads. Detailed steps are described in the main text. (B) Marker proteins for mitochondria (Tom70, Porin, cytochrome b2 [Cyb2], α-ketoglutarate dehydrogenase [Kgd1]), cytosol (hexokinase [Hxk]), ER (Sec61), and vacuole (Nyv1) are probed using Western blot analysis. Total protein load was assessed using TCE. (C) Crude or bead-purified mitochondria are treated with (+) or without (−) 100 μg/ml proteinase K (PK) for 30 min at 4°C. Mitochondrial outer membrane proteins (Tom70 and Porin) and an intermembrane space protein (Cyb2) are probed using Western blot analysis.
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