Isolation of large amounts of highly pure mitochondria for “omics” studies
- Authors: Afanasyeva M.A.1, Ustiugova A.S.1, Golyshev S.A.2, Kopylov A.T.3, Bogolyubova A.V.1, Demin D.E.1,4, Belousov P.V.1, Schwartz A.M.1,4
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Affiliations:
- Engelhardt Institute of Molecular Biology
- Belozersky Institute of Physico-Chemical Biology
- Orekhovich Institute of Biomedical Chemistry
- Moscow Institute of Physics and Technology
- Issue: Vol 83, No 1 (2018)
- Pages: 76-85
- Section: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151573
- DOI: https://doi.org/10.1134/S0006297918010108
- ID: 151573
Cite item
Abstract
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.
About the authors
M. A. Afanasyeva
Engelhardt Institute of Molecular Biology
Author for correspondence.
Email: ama@eimb.ru
Russian Federation, Moscow, 119991
A. S. Ustiugova
Engelhardt Institute of Molecular Biology
Email: ama@eimb.ru
Russian Federation, Moscow, 119991
S. A. Golyshev
Belozersky Institute of Physico-Chemical Biology
Email: ama@eimb.ru
Russian Federation, Moscow, 119234
A. T. Kopylov
Orekhovich Institute of Biomedical Chemistry
Email: ama@eimb.ru
Russian Federation, Moscow, 119121
A. V. Bogolyubova
Engelhardt Institute of Molecular Biology
Email: ama@eimb.ru
Russian Federation, Moscow, 119991
D. E. Demin
Engelhardt Institute of Molecular Biology; Moscow Institute of Physics and Technology
Email: ama@eimb.ru
Russian Federation, Moscow, 119991; Dolgoprudny, Moscow Region, 141701
P. V. Belousov
Engelhardt Institute of Molecular Biology
Email: ama@eimb.ru
Russian Federation, Moscow, 119991
A. M. Schwartz
Engelhardt Institute of Molecular Biology; Moscow Institute of Physics and Technology
Email: ama@eimb.ru
Russian Federation, Moscow, 119991; Dolgoprudny, Moscow Region, 141701