制备枯草芽胞杆菌细胞裂解物和膜

Juri  Niño  Bach; Marc Bramkamp

doi:10.21769/BioProtoc.1025

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Peer-reviewed

Preparation of Bacillus subtilis Cell Lysates and Membranes

制备枯草芽胞杆菌细胞裂解物和膜

JB Juri Niño Bach

MB Marc Bramkamp email

发布: 2014年01月20日第4卷第2期 DOI: 10.21769/BioProtoc.1025 浏览次数: 15850

评审: Fanglian He

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The authors used this protocol in:

Cover of Molecular Microbiology, featuring study using the protocol.

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-M^TM )
Ultra-centrifuge (Beckman Coulter, model: optima^TM 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

English

中文翻译

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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.