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Preparation of Bacillus subtilis Cell Lysates and Membranes
制备枯草芽胞杆菌细胞裂解物和膜

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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

  1. Bacillus subtilis (B. subtilis)
  2. Glycerol (Carl Roth, catalog number: 7530.4 )
  3. Tris (J.T.Baker®, catalog number: 1414 )
  4. NaCl (AppliChem GmbH, catalog number: A3597.5000 )
  5. MgCl2 (Merck KGaA, catalog number: 1.05833.1000 )
  6. Proteinase inhibitor (Roche Diagnostics, catalog number: 0 4693159001 )
  7. DNAse I (Roche Diagnostics, catalog number: 10104159001 )
  8. Lysozyme (Roche Diagnostics, catalog number: 10153516103 )
  9. Casein hydrolysate (Oxoid Limited, catalog number: LP0041 )
  10. Buffer A (see Recipes)
  11. Casein Hydrolysate (CH-medium) (see Recipes)
  12. Solution G (see Recipes)

Equipment

  1. Glass beads (diameter 0.2-0.3 mm) (Sigma-Aldrich, catalog number: G1277 )
  2. French press homogenizer (Glen Mills, French press G-MTM )
  3. Ultra-centrifuge (Beckman Coulter, model: optimaTM XPN-100 )
  4. Ti-70 Rotor (Beckman Coulter)
  5. FastPrep tissue homogenizer (MP Biomedicals, model: 116004500 )
  6. Refrigerated centrifuge (e.g. Beckman Coulter, model: Avanti-J25 )
  7. Acrodisc® syringe filters (a pore size of 0.2 µm) (Pall, catalog number: 4652 )

Procedure

  1. Inoculate B. subtilis to an OD600 of 0.1 in appropriate medium [e.g. CH, lysogeny broth (LB), Spizizen minimal medium (SMM); here we used 50 ml cultures in CH-medium].
  2. Grow cells to respective OD600 (here we used an OD600 of 4, cells growing at 37 °C; 150 rpm in 250 ml flasks with baffles).
  3. Harvest cells by centrifugation (10,000 x g; 15 min; 4 °C).
  4. Discard supernatant and resuspend cells in 1/5 volumes of original culture at 4 °C in pre-cooled Buffer A.
  5. Centrifuge again (10,000 x g; 15 min; 4 °C).
  6. Discard supernatant and resuspend cells in 5-8x times volume of the cell pellet in Buffer A at 4 °C supplemented with proteinase inhibitor and DNase I. Use concentrations as specified by the manufacturer.  
  7.  Disrupt cells
    Note: If cell disruption is critical, the cell suspension can be incubated with lysozyme (50 μg/ml) on ice for 30-120 min prior to cell disruption, check microscopically.
    1. Small cell volumes (1 ml in reaction tubes) can be disrupted in a FastPrep tissue homogenizer for 30 sec at 6.5 m/s with diameter 0.2-0.3 mm glass beads and cooling for 5 min on ice between runs (minimum 5 runs).
    2. Larger cell volumes can be disrupted in a French press homogenizer at 125 MPa and 3-5 passes. Cool sample on ice for 5 min between every pass. Note that only use of a French Press system will yield inside-out vesicles.
  8. Remove cell debris by centrifugation (12,000 x g; 15 min; 4 °C).
  9. Collect the supernatant (cell lysate) and centrifuge it at ≥ 200,000 x g; 4 °C and 60-90 min (e.g. in a Beckman-Coulter® Ti-70 rotor at 45,000 rpm).
    Note: Longer centrifugation time results in a better separation of membranes at high protein concentrations.
  10. The supernatant represents the cytoplasmatic fraction; the pellet contains B. subtilis membranes (lipids, trans-membrane proteins and membrane associated proteins).
    Note: Resuspend the pellet in an appropriate buffer, e.g. Buffer A.

Recipes

  1. Buffer A
    50 mM Tris.HCl (pH 7.5)
    150 mM NaCl
    5 mM MgCl2
    10% Glycerol (v/v)
  2. CH-medium
    50 ml solution G (see below)
    0.05 ml 0.1 M CaCl2
    0.02 ml 1 M MgSO4.7H2O
    0.1 ml 0.0475 M MnSO4
    0.5 ml 2 mg x ml-1 tryptophan
    Sterilize by filtering using Acrodisc® syringe filters; prepare freshly
  3. Solution G
    25.0 g casein hydrolysate
    9.2 g L-glutamic acid
    3.125 g L-alanine
    3.48 g L-asparagine
    3.4 g KH2PO4
    1.34 g NH4Cl
    0.27 g Na2SO4
    0.24 g NH4NO3
    2.45 mg FeCl3.6H2O
    ddH2O 2.35 L and adjust pH to 7.0 by using 10 N NaOH
    Sterilize by autoclaving; stored at 4 °C

Acknowledgments

This protocol is adapted from Bach and Bramkamp (2013).

References

  1. Bach, J. N. and Bramkamp, M. (2013). Flotillins functionally organize the bacterial membrane. Mol Microbiol 88(6): 1205-1217.

简介

每个真核和原核细胞的共同特征是它们表现出质膜。 在枯草芽孢杆菌(<枯草芽孢杆菌)中,大约25%的蛋白质是推定的跨膜或膜相关蛋白。 在这里,我们描述了一种相对简单的方法,通过超离心分离和制备膜和胞质蛋白

材料和试剂

  1. 甘油(Carl Roth,目录号:7530.4)
  2. Tris(J.T.Baker ,目录号:1414)
  3. NaCl(AppliChem GmbH,目录号:A3597.5000)
  4. MgCl 2(Merck KGaA,目录号:1.05833.1000)
  5. 蛋白酶抑制剂(Roche Diagnostics,目录号:04693159001)
  6. DNAse I(Roche Diagnostics,目录号:10104159001)
  7. 溶菌酶(Roche Diagnostics,目录号:10153516103)
  8. 酪蛋白水解物(Oxoid Limited,目录号:LP0041)
  9. 缓冲区A(参见配方)
  10. 酪蛋白水解物(CH-培养基)(参见配方)
  11. 解决方案G(参见配方)

设备

  1. 玻璃珠(直径0.2-0.3mm)(Sigma-Aldrich,目录号:G1277)
  2. 法式压力匀浆器(Glen Mills,French press G-M )
  3. 超离心机(Beckman Coulter,型号:optima TM XPN-100)
  4. Ti-70转子(Beckman Coulter)
  5. FastPrep组织匀浆器(MP Biomedicals,型号:116004500)
  6. 冷冻离心机(例如:Beckman Coulter,型号:Avanti-J25)
  7. Acrodisc 注射器过滤器(孔径为0.2μm)(Pall,目录号:4652)

程序

  1. 接种 B。 (LB),Spizizen基本培养基(SMM)中,在适当的培养基[例如CH]中培养至OD 600为0.1。 这里我们使用CH培养基中的50ml培养物]
  2. 将细胞生长至相应的OD 600(这里我们使用OD 600为4,在37℃下生长的细胞;在具有挡板的250ml烧瓶中以150rpm培养)。< br/>
  3. 通过离心收集细胞(10,000×g; 15分钟; 4℃)
  4. 弃去上清液,并在4℃下在预冷的缓冲液A中的1/5体积的原始培养物中重悬细胞
  5. 再次离心(10,000×g; 15分钟; 4℃)
  6. 弃去上清液,并在补充有蛋白酶抑制剂和DNA酶I的4℃下在缓冲液A中的5-8x体积的细胞沉淀中重悬细胞。使用制造商指定的浓度。  
  7.  中断单元格
    注意:如果细胞破碎是关键的,细胞悬浮液可以与溶菌酶(50μg/ml)在冰上温育30-120分钟,然后细胞破碎,显微镜检查。
    1. 小细胞体积(反应管中1ml)可以在FastPrep组织匀浆器中以6.5m/s用直径0.2-0.3mm玻璃珠破碎30秒,并在运行之间在冰上冷却5分钟(最小5 运行)。
    2. 更大的细胞体积可以在French Press匀浆器中在125MPa和3-5次通过下破碎。 将样品在冰上冷却5分钟。 请注意,只有使用法语Press系统才会生成内向外的囊泡。
  8. 通过离心(12,000×g ; 15分钟; 4℃)除去细胞碎片
  9. 收集上清液(细胞裂解物),并以≥200,000×g离心; 4℃和60-90分钟(例如在Beckman-Coulter Ti-70转子中以45,000rpm)。
    注意:离心时间越长,蛋白质浓度越高,膜的分离越好。
  10. 上清液表示细胞质部分; 小球包含 B。 枯草芽孢杆菌膜(脂质,跨膜蛋白和膜相关蛋白)。
    注意:将沉淀重悬于适当的缓冲液中,例如, 缓冲区A。

食谱

  1. 缓冲区A
    50mM Tris·HCl(pH7.5) 150mM NaCl 5mM MgCl 2/
    10%甘油(v/v)
  2. CH-medium
    50ml溶液G(见下文)
    0.05ml 0.1M CaCl 2·h/v 0.02ml 1M MgSO 4。7H 2 O 2 0.1ml 0.0475M MnSO 4
    0.5ml 2mg xml 色氨酸 注射器过滤器过滤灭菌;使用Acrodisc 新鲜准备
  3. 解决方案G
    25.0g酪蛋白水解产物 9.2g L-谷氨酸 3.125g L-丙氨酸 3.48g L-天冬酰胺 3.4g KH 2 PO 4 sub/
    1.34g NH 4 Cl
    0.27g Na 2 SO 4。
    0.24g NH 4 NO 3 sub。 2.45mg FeCl 3 。 6H 2 O ddH 2 O 2.35L,并用10N NaOH调节pH至7.0 高压灭菌; 储存在4℃下

致谢

该协议改编自Bach和Bramkamp(2013)。

参考文献

  1. Bach,J.N.和Bramkamp,M。(2013)。 Flotillins功能性组织细菌膜。 Mol Microbiol 88 (6):1205-1217。
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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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