Enrichment of Golgi membranes from HeLa cells by sucrose gradient ultracentrifugation
通过蔗糖梯度超速离心法从HeLa细胞中富集高尔基体膜
发布: 2013年09月20日第3卷第18期 DOI: 10.21769/BioProtoc.906 浏览次数: 15863
评审: Lin FangFanglian HeHui Zhu
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参见作者原研究论文
The authors used this protocol in:
Dec 2012
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Abstract
This is a protocol to extract intact Golgi Membranes from HeLa cells using sucrose gradient centrifugation. This extraction is very useful for several applications including pull-down of Golgi membrane proteins, electron microscopy and reconstitution of protein transport into an isolated system. Protocol adapted from Balch et al. (1984).
Keywords: Golgi membranes (高尔基体膜)
HeLa cells (HeLa细胞)
Sucrose gradient cenrifugation (蔗糖梯度cenrifugation)
Materials and Reagents
- HeLa cells (ATTC, Wesel, Germany)
- PBS
- 1 M Tris (pH 7.4)
- 100 mM EDTA
- Trypan Blue
- Protease inhibitor cocktail tablets (Roche, catalog number: 11836153001 )
- Breaking buffer (BB) (see Recipes)
- 29% (w/w) sucrose (see Recipes)
- 35% (w/w) sucrose (see Recipes)
- 62% (w/w) sucrose (see Recipes)
Equipment
- Cell scrapers
- Cell homogenizer (EMBL cell cracker) (EMBLEM Technology Transfer, Heidelberg)
- Cell culture microscope
- Ultracentrifuge (Beckman Coulter, model: Optima L-100K or equivalent)
- Refractometer
- SW40Ti rotor
- Centrifuge tubes
- 1 ml syringe with 20/21 G needle
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Readers should cite both the Bio-protocol article and the original research article where this protocol was used:
- Galen, J. V. and Blume, J. V. (2013). Enrichment of Golgi membranes from HeLa cells by sucrose gradient ultracentrifugation. Bio-protocol 3(18): e906. DOI: 10.21769/BioProtoc.906.
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von Blume, J., Alleaume, A.-M., Kienzle, C., Carreras-Sureda, A., Valverde, M. and Malhotra, V. (2012). Cab45 is required for Ca2+-dependent secretory cargo sorting at the trans-Golgi network. J Cell Biol 199(7): 1057-1066.
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分类
细胞生物学 > 细胞器分离 > 高尔基体
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