Preparation of Bacillus subtilis Cell Lysates and Membranes
制备枯草芽胞杆菌细胞裂解物和膜
发布: 2014年01月20日第4卷第2期 DOI: 10.21769/BioProtoc.1025 浏览次数: 16689
评审: Fanglian He
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参见作者原研究论文
The authors used this protocol in:
Jun 2013
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Abstract
A common feature of every eukaryotic and prokaryotic cell is that they exhibit a plasma membrane. In Bacillus subtilis (B. subtilis) roughly 25% of all proteins are putative trans- or membrane associated proteins. Here we describe a relatively simple method to separate and prepare membrane and cytosolic proteins by ultra-centrifugation.
Materials and Reagents
- Bacillus subtilis (B. subtilis)
- Glycerol (Carl Roth, catalog number: 7530.4 )
- Tris (J.T.Baker®, catalog number: 1414 )
- NaCl (AppliChem GmbH, catalog number: A3597.5000 )
- MgCl2 (Merck KGaA, catalog number: 1.05833.1000 )
- Proteinase inhibitor (Roche Diagnostics, catalog number: 0 4693159001 )
- DNAse I (Roche Diagnostics, catalog number: 10104159001 )
- Lysozyme (Roche Diagnostics, catalog number: 10153516103 )
- Casein hydrolysate (Oxoid Limited, catalog number: LP0041 )
- Buffer A (see Recipes)
- Casein Hydrolysate (CH-medium) (see Recipes)
- Solution G (see Recipes)
Equipment
- Glass beads (diameter 0.2-0.3 mm) (Sigma-Aldrich, catalog number: G1277 )
- French press homogenizer (Glen Mills, French press G-MTM )
- Ultra-centrifuge (Beckman Coulter, model: optimaTM XPN-100 )
- Ti-70 Rotor (Beckman Coulter)
- FastPrep tissue homogenizer (MP Biomedicals, model: 116004500 )
- Refrigerated centrifuge (e.g. Beckman Coulter, model: Avanti-J25 )
- Acrodisc® syringe filters (a pore size of 0.2 µm) (Pall, catalog number: 4652 )
Procedure
文章信息
版权信息
© 2014 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Bach, J. N. and Bramkamp, M. (2014). Preparation of Bacillus subtilis Cell Lysates and Membranes. Bio-protocol 4(2): e1025. DOI: 10.21769/BioProtoc.1025.
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分类
细胞生物学 > 细胞器分离 > 细胞膜
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