Isolation of large amounts of highly pure mitochondria for “omics” studies
- Авторы: Afanasyeva M.1, Ustiugova A.1, Golyshev S.2, Kopylov A.3, Bogolyubova A.1, Demin D.1,4, Belousov P.1, Schwartz A.1,4
-
Учреждения:
- Engelhardt Institute of Molecular Biology
- Belozersky Institute of Physico-Chemical Biology
- Orekhovich Institute of Biomedical Chemistry
- Moscow Institute of Physics and Technology
- Выпуск: Том 83, № 1 (2018)
- Страницы: 76-85
- Раздел: Article
- URL: https://journals.rcsi.science/0006-2979/article/view/151573
- DOI: https://doi.org/10.1134/S0006297918010108
- ID: 151573
Цитировать
Аннотация
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.
Ключевые слова
Об авторах
M. Afanasyeva
Engelhardt Institute of Molecular Biology
Автор, ответственный за переписку.
Email: ama@eimb.ru
Россия, Moscow, 119991
A. Ustiugova
Engelhardt Institute of Molecular Biology
Email: ama@eimb.ru
Россия, Moscow, 119991
S. Golyshev
Belozersky Institute of Physico-Chemical Biology
Email: ama@eimb.ru
Россия, Moscow, 119234
A. Kopylov
Orekhovich Institute of Biomedical Chemistry
Email: ama@eimb.ru
Россия, Moscow, 119121
A. Bogolyubova
Engelhardt Institute of Molecular Biology
Email: ama@eimb.ru
Россия, Moscow, 119991
D. Demin
Engelhardt Institute of Molecular Biology; Moscow Institute of Physics and Technology
Email: ama@eimb.ru
Россия, Moscow, 119991; Dolgoprudny, Moscow Region, 141701
P. Belousov
Engelhardt Institute of Molecular Biology
Email: ama@eimb.ru
Россия, Moscow, 119991
A. Schwartz
Engelhardt Institute of Molecular Biology; Moscow Institute of Physics and Technology
Email: ama@eimb.ru
Россия, Moscow, 119991; Dolgoprudny, Moscow Region, 141701