Subcellular Fractionation of Cultured Human Cell Lines
人源细胞系的亚细胞分离
发布: 2013年05月05日第3卷第9期 DOI: 10.21769/BioProtoc.754 浏览次数: 51441
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The authors used this protocol in:
Aug 2012
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Abstract
Subcellular localization is crucial for the proper functioning of a protein. Deregulation of subcellular localization may lead to pathological consequences and result in diseases like cancer. Immuno-fluorescent staining and subcellular fractionation can be used to determine localization of a protein. Here we discuss a protocol to separate the nuclear, cytosolic, and membrane fractions of cultured human cell lines using a centrifuge and ultracentrifuge. The membrane fraction contains plasma membranes and ER-golgi membranes, but no mitochondria or nuclear structures. The fractions can be further analyzed using Western blotting. This protocol is based on that from Dr. Richard Patten at Abcam, and was modified and utilized in a publication by Huang et al. (2012).
Keywords: Nuclear (细胞核)
Cytosolic (细胞溶质的)
Membrane (膜)
Centrifugation (离心)
Fractionation (分馏)
Materials and Reagents
- Sucrose
- HEPES
- Potassium chloride (KCl)
- Magnesium chloride (MgCl2)
- Ethylene diamine tetraacetic acid (EDTA)
- Ethylene glycol tetraacetic acid (EGTA)
- Dithiothreitol (DTT)
- Tris (Affymetrix-USB, catalog number: 75825 )
- Sodium chloride (NaCl) (Sigma-Aldrich, catalog number: 13565 )
- Nonidet P40 substitute (NP40)
- Sodium deoxycholate
- Glycerol
- Sodium dodecyl sulfate (SDS)
- Protease inhibitor (PI) cocktails (F. Hoffmann-La Roche, catalog number: 11836145001 )
- Methanol
- Acetic acid
- Brilliant Blue R (Affymetrix, catalog number: 32826 )
- Phosphate buffered saline (PBS)
- Histone H3 antibody (Cell Signaling Technology, catalog number: 9715 )
- Alpha-tubulin antibody (GeneTex, catalog number: GTX108784 )
- Cell scraper (BD Biosciences, Falcon®, catalog number: 353086 )
- Subcellular fractionation buffer (SF buffer) (see Recipes)
- Nuclear Lysis buffer (NL buffer) (see Recipes)
- Brilliant Blue R staining solution and destaining solution (see Recipes)
Equipment
- 4 °C Microcentrifuge (Eppendorf, catalog number: 5415R );
- Ultracentrifuge (Beckman Coulter, model number: Optima TLX )
- Optional: Sonicator (Sonics, model number: VC505 )
- Whatman filter paper
- 37 °C incubator
- 1.5 ml Eppendorf microtubes
- Tube roller (Maplelab-scientific, model number: MTR-1D )
Procedure
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文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Yu, Z., Huang, Z. and Lung, M. L. (2013). Subcellular Fractionation of Cultured Human Cell Lines. Bio-protocol 3(9): e754. DOI: 10.21769/BioProtoc.754.
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分类
癌症生物学 > 通用技术 > 生物化学试验 > 蛋白质分析
生物化学 > 蛋白质 > 分离和纯化
细胞生物学 > 细胞器分离 > 分级分离
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