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Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles
卡他莫拉菌外膜蛋白图谱分析   

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Abstract

Phenotypes observed for certain Moraxella catarrhalis wild-type strains or mutants may be caused by a variety of factors including alteration of outer membrane protein composition. Examination of the outer membrane protein profiles may be a valuable tool to identify changes in outer membrane compositions of these strains. Here we describe a method to isolate and analyse M. catarrhalis fractions highly enriched for membrane proteins.

Keywords: Moraxella catarrhalis(卡他莫拉菌), Outer membrane vesicles(外膜囊泡), Pathogenesis(发病机制), Virulence(毒力)

Materials and Reagents

  1. Brain heart infusion (BHI) (Becton Dickinson and Company, catalog number: 237500 ) broth and BHI agar plates
  2. Antibiotics
    e.g. spectinomycin or kanamycin (Merck KgaA, Calbiochem, catalog numbers: 567570-10 and 420311-5 )
  3. PBS
  4. Bovine skin gelatin (Sigma-Aldrich, catalog number: G9382-100G )
  5. ReadyPrep Protein Extraction Kit (membrane I) (Bio-Rad, catalog number: 1632088 )
  6. Glass beads, acid-washed (150-212 μm) (Sigma-Aldrich, catalog number: G1145 )
  7. 2D-Quant Kit (General Electric Company, catalog number: 80-6483-56 )
  8. Mini-protean TGX precast gels, 4-15% (Bio-Rad, catalog number: 456-1083 )
  9. Colloidal Coomassie staining (Pink et al., 2012)

Equipment

  1. CO2 incubator
  2. Benchtop Incubator Shaker
  3. Centrifuge
  4. TissueLyser LT (QIAGEN, model: 85600 )

Procedure

  1. M. catarrhalis strains were inoculated on brain heart infusion (BHI) plates (supplemented with antibiotics when appropriate), and grown overnight at 37 °C in an atmosphere containing 5% CO2.
  2. Bacteria were harvested from plate and resuspended in PBS supplemented with 0.15% gelatin (PBS-G). This suspension was used to inoculate BHI broth to an OD620 nm of ~ 0.05 and grown at 37 °C at 200-250 rpm until OD620 nm of 1.0 to 1.2 (mid-log). Use of different growth media is possible, but may affect outer membrane protein profiles. Always use the same growth media when comparing outer membrane protein profiles of different strains.
  3. Subsequently, bacteria were harvested by centrifugation for 10 minutes at 3,200 x g.
  4. Outer membranes were isolated using the ReadyPrep Membrane I kit according to manufacturer’s instructions.
  5. During the lysis procedure, 50 mg acid-washed glass beads (150-212 μm) were added for homogenization with the TissueLyser LT. The TissueLyser was operated 5 times for 1 minute at 50 Hz.
  6. Suspensions were chilled on ice for 1 minute between every TissueLyser step.
  7. Protein quantification was performed using the 2D-Quant Kit according to manufacturer’s instructions.
  8. Five microgram of outer membrane preparations were separated on a 4-15% TGX gel and analyzed by colloidal Coomassie staining (Pink et al., 2010).

Acknowledgments

This protocol was published in: de Vries et al. (2013). This study was financially supported by Vienna Spot of Excellence (VSOE) grant (ID337956).

References

  1. de Vries, S. P., Burghout, P., Langereis, J. D., Zomer, A., Hermans, P. W. and Bootsma, H. J. (2013). Genetic requirements for Moraxella catarrhalis growth under iron-limiting conditions. Mol Microbiol 87(1): 14-29.    
  2. Pink, M., Verma, N., Rettenmeier, A. W. and Schmitz-Spanke, S. (2010). CBB staining protocol with higher sensitivity and mass spectrometric compatibility. Electrophoresis 31(4): 593-598.   

简介

对某些粘膜炎莫拉菌的野生型菌株或突变体观察到的表型可能由多种因素引起,包括外膜蛋白组成的改变。 外膜蛋白质谱的检查可以是鉴定这些菌株的外膜组成的变化的有价值的工具。 在这里我们描述一种方法来隔离和分析。 粘膜炎的部分高度富集膜蛋白。

关键字:卡他莫拉菌, 外膜囊泡, 发病机制, 毒力

材料和试剂

  1. 脑心浸液(BHI)(Becton Dickinson and Company,目录号:237500)肉汤和BHI琼脂平板
  2. 抗生素
    例如壮观霉素或卡那霉素(Merck KgaA,Calbiochem,目录号:567570-10和420311-5)
  3. PBS
  4. 牛皮明胶(Sigma-Aldrich,目录号:G9382-100G)
  5. ReadyPrep蛋白提取试剂盒(膜I)(Bio-Rad,目录号:1632088)
  6. 酸洗(150-212μm)(Sigma-Aldrich,目录号:G1145)的玻璃珠
  7. 2D-Quant Kit(通用电气公司,目录号:80-6483-56)
  8. Mini-protean TGX预制凝胶,4-15%(Bio-Rad,目录号:456-1083)
  9. 胶体考马斯染色(Pink等人,2012)

设备

  1. CO <2>孵化器
  2. 台式孵化器
  3. 离心机
  4. TissueLyser LT(QIAGEN,型号:85600)

程序

  1. M。 卡那霉素菌株接种在脑心浸液(BHI)平板(当合适时补充有抗生素)中,并在37℃下在含有5%CO 2的气氛中生长过夜。
  2. 从板中收获细菌并重悬于补充有0.15%明胶(PBS-G)的PBS中。将该悬浮液用于接种BHI肉汤至〜550nm的OD 620nm,并在37℃下以200-250rpm生长,直到OD 620nm为1.0至1.2中日志)。使用不同的生长培养基是可能的,但可能影响外膜蛋白质谱。当比较不同菌株的外膜蛋白谱时,始终使用相同的生长培养基。
  3. 随后,通过以3,200×g离心10分钟收获细菌。
  4. 使用ReadyPrep Membrane I试剂盒根据制造商的说明书分离外膜。
  5. 在裂解程序期间,加入50mg酸洗玻璃珠(150-212μm)以与TissueLyser LT均化。 TissueLyser在50Hz下操作5次1分钟。
  6. 将悬浮液在冰上在每个TissueLyser步骤之间冷冻1分钟
  7. 使用2D-Quant试剂盒根据制造商的说明书进行蛋白质定量。
  8. 在4-15%TGX凝胶上分离5微克外膜制剂,并通过胶体考马斯染色(Pink等人,2010)进行分析。

致谢

该协议发表于:de Vries et al。(2013)。 本研究得到了维也纳卓越奖(VSOE)资助(ID337956)的资助。

参考文献

  1. de Vries,S.P.,Burghout,P.,Langereis,J.D.,Zomer,A.,Hermans,P.W.和Bootsma,H.J。(2013)。 在铁限制条件下对粘膜炎莫拉氏菌生长的遗传学要求。 Mol Microbiol 87(1):14-29。    
  2. Pink,M.,Verma,N.,Rettenmeier,A.W.and Schmitz-Spanke,S。(2010)。 CBB染色方案,具有更高的灵敏度和质谱兼容性。 em> 31(4):593-598。   
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Vries, S. P. and Bootsma, H. J. (2013). Analysis of Moraxella catarrhalis Outer Membrane Protein Profiles. Bio-protocol 3(21): e957. DOI: 10.21769/BioProtoc.957.
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