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Biochemistry (Moscow). Papers in Press. Published on November 27, 2017 as Manuscript BM17-461.

Isolation of Large Amounts of Highly Pure Mitochondria for “Omics” Studies

M. A. Afanasyeva1*, A. S. Ustiugova1, S. A. Golyshev2, A. T. Kopylov3, A. V. Bogolyubova1, D. E. Demin1,4, P. V. Belousov1, and A. M. Schwartz1,4

1Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, 119991 Moscow, Russia; E-mail: ama@eimb.ru

2Lomonosov Moscow State University, Belozersky Institute of Physico-Chemical Biology, 119234 Moscow, Russia

3Institute of Biomedical Chemistry, Russian Academy of Medical Sciences, 119121 Moscow, Russia

4Moscow Institute of Physics and Technology, 141701 Dolgoprudny, Moscow Region, Russia

*To whom correspondence should be addressed.

Received October 13, 2017; Revision received October 27, 2017
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.
KEY WORDS: mitochondria isolation, proteomics, two-dimensional gel electrophoresis