Isolation of Large Amounts of Highly Pure Mitochondria for "Omics" Studies.
Biochemistry (Mosc)
; 83(1): 76-85, 2018 Jan.
Article
em En
| MEDLINE
| ID: mdl-29534672
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.
Texto completo:
1
Base de dados:
MEDLINE
Assunto principal:
Fracionamento Celular
/
Proteínas Mitocondriais
/
Proteômica
/
Mitocôndrias
Limite:
Humans
Idioma:
En
Ano de publicação:
2018
Tipo de documento:
Article