Preparation of Pneumococcal Proteins for Western Blot Analysis
制备肺炎球菌蛋白用于蛋白印迹检测
发布: 2013年07月05日第3卷第13期 DOI: 10.21769/BioProtoc.807 浏览次数: 9773
评审: Fanglian He
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参见作者原研究论文
The authors used this protocol in:
Jan 2013
Abstract
This protocol was developed in a study aimed to determine the cellular localization of the lysin of pneumococcal phage SV1 (Frias et al., 2013). We obtained proteins from the surface of Streptococcus pneumoniae by elution with choline or those secreted to the medium. The analysis by Western blot of these fractions allowed us to demonstrate that the phage lysin localizes to the cell wall, associating with choline residues in the teichoic acids. Hence, protein extracts can be used to determine the localization of uncharacterized proteins and can also be useful for other biochemical analyses such as protein identification. This protocol can be easily adapted to different pneumococcal strains and growth conditions and it is well suited to isolate other proteins of interest.
Materials and Reagents
- Pneumococcal cells
- Mitomycin C (MitC) (0.1 μg/ml) (Sigma-Aldrich, catalog number: M0503 )
- 1x PBS (10x PBS pH 7.2) (Life Technologies, Gibco®/Invitrogen®, catalog number: 70013-016 )
- 50 mM Tris pH 7.5
- 2% (w/v) Choline chloride in 1x PBS (Sigma-Aldrich, catalog number: C7527 )
- NaCl (AppliChem, catalog number: A46615000 )
- Tris-HCl (Bio-Rad Laboratories, catalog number: 161-0799 )
- Glycerol (AppliChem, catalog number: A2364, 5000 )
- SDS (Bio-Rad Laboratories, catalog number: 161-0416 )
- β-mercaptoethanol (Sigma-Aldrich, catalog number: M3148 )
- Bromophenol blue (Bio-Rad Laboratories, catalog number: 161-0404 )
- Casamino acids
- L-Tryptophan
- L-Cysteine.HCl
- Glutamine
- Adenosine
- Uridine
- Nicotinic acid
- Pyridoxine
- Ca-pantothenate
- Thiamine-HCl
- Riboflavin
- Biotin
- Asparagine
- Casein-based semisynthetic medium C+Y (Lacks and Hotchkiss, 1960) (see Recipes)
- Loading buffer 5x (see Recipes)
- C+Y with 2% choline chloride (see Recipes)
Equipment
- Water bath at 37 °C to grow bacterial cultures
- Cell density meter (Biochrom WPA CO8000 Cell Density Meter) (Biochrom, catalog number: 80-3000-45 )
- Centrifuge
- 0.2 μm low-binding-protein membrane (DISMIC-03CP) (Advantec, catalog number: 03CP020AS )
- 0.22 μm membrane filter (Frilabo, catalog number: 1520012 )
- Amicon Ultra-15 centrifugal filter unit, cut-off 10 kDa (Merck Millipore, catalog number: UFC901024 )
- Western blot equipment
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Frias, M. J., Melo-Cristino, J. and Ramirez, M. (2013). Preparation of Pneumococcal Proteins for Western Blot Analysis. Bio-protocol 3(13): e807. DOI: 10.21769/BioProtoc.807.
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分类
微生物学 > 微生物生物化学 > 蛋白质
生物化学 > 蛋白质 > 免疫检测
生物化学 > 蛋白质 > 分离和纯化
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