Separation of the Inner and Outer Mitochondrial Membrane in HeLa Cells
HeLa细胞中线粒体内外膜的分离
发布: 2014年11月20日第4卷第22期 DOI: 10.21769/BioProtoc.1299 浏览次数: 29660
评审: Arsalan DaudiVarpu MarjomakiJyotiska Chaudhuri
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参见作者原研究论文
The authors used this protocol in:
May 2014
Abstract
Mitochondria are organelles that have important functions in oxidative phosphorylation, fatty acid oxidation and apoptosis signaling. They have two distinct membranes, outer membrane (OM) and inner membrane (IM). IM contains respiratory chain complexes that produce ATP. IM is rich in cardiolipin, a specific phospholipid reportedly having a critical role for organizing super-complex formation of respiratory chain complexes. IM abundant in cardiolipin exhibits resistance to extraction by digitonin (a non-ionic detergent), whereas the detergent easily lyses OM. Therefore, digitonin is useful to separate mitoplast (IM plus matrix) and OM from mitochondria. Here, we describe a method to isolate mitochondria from HeLa cells, and a method to isolate mitochondrial outer membrane proteins and inner membrane proteins by using digitonin. This method is applicable also to other types of cultured cells such as COS-7.
Keywords: Mitochondria (线粒体)
Membrane (膜)
Isolation (隔离)
Inner (内部的)
Outer (外面的)
Materials and Reagents
- HeLa cells (80-100% confluent) pre-cultured in 8 to 12 pieces of dishes (diameter 10-cm)
- PBS (-) (without Ca2+ and Mg2+)
- EDTA
- Mannitol
- Sucrose
- HEPES
- EGTA
- Complete Mini EDTA-free (Roche Diagnostics, catalog number: 11 836 170 001 )
- Digitonin (Wako Pure Chemical Industries, catalog number: 040-02123 )
- Trichloroacetic acid (optional)
- Mitochondria isolation buffer (MTiso-buffer) (see Recipes)
- Digitonin stock solution (see Recipes)
- Digitonin buffer (see Recipes)
Equipment
- Culture dishes
- Aspirator
- Cell scraper (e.g. Rubber policeman)
- Dounce homogenizer (Glass 7 ml Dounce Tissue Grinder) (WHEATON, catalog number: 357542 )
- 50 ml-tube
- Phase-contrast microscope (upright-type, x100 ~ x400) (for confirming disruption efficiency of the cells)
- Centrifuges [for centrifugation at 500 x g (swing rotor) and 10,000 x g (angle rotor)]
- Micro tube mixer (e.g. TOMY MT-400 , Digital Biology, catalog number: MT-400) or vortex mixer
- Ultracentrifuge (e.g. Hitachi, catalog number: CS100 ) (for centrifugation at 100,000 x g (angle rotor)
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Nishimura, N. and Yano, M. (2014). Separation of the Inner and Outer Mitochondrial Membrane in HeLa Cells. Bio-protocol 4(22): e1299. DOI: 10.21769/BioProtoc.1299.
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分类
细胞生物学 > 细胞器分离 > 线粒体
细胞生物学 > 细胞信号传导 > 发育
生物化学 > 蛋白质 > 分离和纯化
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