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Surface Polysaccharide Extraction and Quantification
表面多糖提取和量化   

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Abstract

Gram-negative bacterial cells possess two membranes - the inner cytoplasmic membrane and the outer membrane. The two membranes are distinct in their composition; the inner membrane is composed of a phospholipid bilayer, whereas the outer membrane (OM) is composed of an asymmetrical bilayer, with the outer leaflet containing lipopolysaccharide (LPS) (Raetz and Whitfield, 2002). Surface polysaccharides, such as LPS O-antigen, or capsular polysaccharide, are often tightly associated with the OM (Whitfield, 2006). This tight association can be used to generate a rough quantification of surface polysaccharides of Gram-negative bacterial cells, as the OM can easily be dissociated from cells without associated cell lysis (Brimacombe et al., 2013). The following method describes how to quickly extract and quantify OM-associated polysaccharides.

Keywords: Polysaccharide(多糖), Capsule(胶囊), Quantification(量化), Outer-membrane(外膜)

Materials and Reagents

  1. Culture of bacterial cells (This procedure works only for Gram-negative bacteria, for example Escherichia coli, Pseudomonas aeurginosa, or Rhodobacter capsulatus. The outer membrane, specifically LPS, is essential for this procedure to work)
  2. 50 mM sodium chloride (NaCl) dissolved in deionized H2O
  3. 50 mM ethylenediaminetetraacetic acid (EDTA) (EMD Millipore, catalog number: 324503 )
  4. Phenol (Fisher Scientific, catalog number: A92-100 )
  5. 93% sulfuric acid (Avantor Performance Materials, catalog number: 2900-10 )
  6. Carbohydrate stock solution (see Recipes)

Equipment

  1. Microcentrifuge
  2. Microfuge tubes (ESBE, catalog number: ESB-ES00507C )
  3. Spectrophotometer
  4. Cuvettes
  5. Glass test tubes
  6. Glass pipettes

Procedure

  1. Extraction of surface polysaccharides from Gram-negative bacteria
    1. Grow bacteria to desired growth phase (generally stationary phase) in desired growth medium.
      Note: Growth medium may affect surface polysaccharide levels, so the same media should be used for all experiments if possible.
    2. Measure OD650 of culture; dilute to < 1 OD if necessary to get an accurate measurement.
    3. Normalize cultures to OD650 of 2.0 (or to maximum OD that bacterial culture will grow to if it is less than 2.0).
    4. Harvest 1 ml of each normalized culture by centrifugation at 14,500 x g for 5 minutes in a microcentrifuge.
    5. Carefully remove supernatant with a pipette, discard tip.
    6. Wash cells by re-suspending in 1 ml of 50 mM NaCl, pellet by centrifugation at 14, 500 x g for 5 minutes, remove supernatant.
    7. Repeat step A6 four additional times (5 total washes).
    8. Re-suspend cells in 1 ml of 50 mM EDTA, and incubate at 37 °C for 60 minutes (EDTA causes LPS to dissociate, thus releasing the OM from cells).
    9. Pellet cells by centrifugation at 14,500 x g for 5 minutes, carefully remove supernatant and transfer to fresh microfuge tube (supernatant contains all surface polysaccharides, including LPS, capsule etc.).

  2. Quantification of surface polysaccharides
    1. Prepare carbohydrate standards by diluting carbohydrate stock solution into 1 ml aliquots of: 0, 30, 60, 90, and 120 μg/ml of carbohydrate (e.g. 970 μl of dH2O + 30 μl of 1 mg/ml stock solution to generate a 30 μg/ml standard).
    2. Prepare clean, acid washed glass test tubes (for a suggested protocol, see Reference 4). Pipette 200 μl of standards, a 200 μl control of 50 mM EDTA, and all test samples into separate tubes.
    3. Move to fume hood.
    4. Add 200 μl of 5% phenol to all tubes, mix well by shaking.
    5. Add 1 ml of 93% sulfuric acid; mix well by swirling (use caution).
    6. Allow colour to develop for 10 minutes at room temperature (reaction should turn yellow; intensity depends on carbohydrate concentration). Additional mixing by gentle swirling every 2-3 minutes may help reaction proceed faster.
    7. Measure OD490 of all reactions in a spectrophotometer; concentration of carbohydrates can then be calculated from the standard curve.
      Note: If necessary, dilute reactions in dH2O to get accurate spectrophotometer readings.

Representative Data

Recipes

  1. Carbohydrate stock solution
    50:50 mixture of 0.5 mg/ml each of sucrose and fructose
    Final concentration of 1 mg/ml carbohydrate (molecular biology grade recommended)

Acknowledgments

The development of this protocol was funded by a grant to J.T.B. from the Canadian Institutes of Health Research.

References

  1. Brimacombe, C. A., Stevens, A., Jun, D., Mercer, R., Lang, A. S. and Beatty, J. T. (2013). Quorum-sensing regulation of a capsular polysaccharide receptor for the Rhodobacter capsulatus gene transfer agent (RcGTA). Mol Microbiol 87(4): 802-817. 
  2. http://openwetware.org/images/9/9e/GLASSWARE_CLEANING_PROCEDURES.pdf
  3. Raetz, C. R. and Whitfield, C. (2002). Lipopolysaccharide endotoxins. Annu Rev Biochem 71: 635-700.
  4. Whitfield, C. (2006). Biosynthesis and assembly of capsular polysaccharides in Escherichia coli. Annu Rev Biochem 75: 39-68.

简介

革兰氏阴性细菌细胞具有两个膜 - 内部细胞质膜和外部膜。 两种膜的组成是不同的; 内膜由磷脂双层组成,而外膜(OM)由不对称双层组成,外层含有脂多糖(LPS)(Raetz和Whitfield,2002)。 表面多糖,如LPS O-抗原或荚膜多糖,通常与OM紧密相关(Whitfield,2006)。 这种紧密关联可以用于产生革兰氏阴性细菌细胞的表面多糖的粗略定量,因为OM可以容易地从没有相关细胞裂解的细胞中解离(Brimacombe等人,2013)。 以下方法描述如何快速提取和定量OM相关多糖。

关键字:多糖, 胶囊, 量化, 外膜

材料和试剂

  1. 细菌细胞的培养(此程序仅适用于革兰氏阴性细菌,例如大肠杆菌,假单胞菌属aeurginosa或红色杆菌 膜,特别是LPS,对于该程序是必不可少的)
  2. 溶解在去离子H 2 O中的50mM氯化钠(NaCl)
  3. 50mM乙二胺四乙酸(EDTA)(EMD Millipore,目录号:324503)
  4. 苯酚(Fisher Scientific,目录号:A92-100)
  5. 93%硫酸(Avantor Performance Materials,目录号:2900-10)
  6. 碳水化合物储备溶液(见配方)

设备

  1. 微量离心机
  2. 微量离心管(ESBE,目录号:ESB-ES00507C)
  3. 分光光度计
  4. 比色杯
  5. 玻璃试管
  6. 玻璃吸管

程序

  1. 从革兰氏阴性细菌中提取表面多糖
    1. 在所需的生长培养基中将细菌生长至所需的生长期(通常为固定期) 注意:生长培养基可能影响表面多糖水平,因此,如果可能,应该对所有实验使用相同的培养基。
    2. 测量培养物的OD 650; 稀释至< 1 OD,如有必要可获得准确的测量
    3. 将培养物标准化至2.0的OD 650(或者如果小于2.0,则为细菌培养物将生长的最大OD)。
    4. 在微量离心机中以14,500×g离心5分钟,收获1ml的每种标准化培养物。
    5. 用移液管小心取出上清液,丢弃尖端
    6. 通过重悬于1ml的50mM NaCl中来洗涤细胞,通过在14,500×g离心5分钟沉淀沉淀,除去上清液。
    7. 重复步骤A6四次(总共5次洗涤)。
    8. 将细胞重新悬浮在1ml的50mM EDTA中,并在37℃下孵育60分钟(EDTA使LPS解离,从而从细胞释放OM)。
    9. 通过在14,500×g离心5分钟沉淀细胞,小心地除去上清液并转移到新鲜微量离心管(上清液含有所有表面多糖,包括LPS,胶囊等)。< br />
  2. 表面多糖的定量
    1. 通过将碳水化合物储备溶液稀释成1ml的等分试样来制备碳水化合物标准品:0,30,60,90和120μg/ml的碳水化合物(例如970μl的dH 2 < O +30μl1mg/ml储备溶液以产生30μg/ml标准品)
    2. 准备清洁,酸洗玻璃试管(建议方案,参见参考文献4)。移取200微升标准品,200微升控制50毫米EDTA,所有测试样品分开管
    3. 移动到通风橱。
    4. 向所有管中加入200μl5%苯酚,通过摇动充分混合
    5. 加入1ml 93%硫酸; 通过旋转混合(使用小心)
    6. 允许颜色在室温下显色10分钟(反应应该变黄;强度取决于碳水化合物浓度)。 通过每2-3分钟轻轻涡旋进行另外的混合可以帮助反应更快地进行。
    7. 在分光光度计中测量所有反应的OD 490; 然后可以从标准曲线计算碳水化合物的浓度。
      注意:如果必要,在dH 2 O中稀释反应以获得精确的分光光度计读数。

代表数据

食谱

  1. 碳水化合物储备液
    50:50的蔗糖和果糖各0.5mg/ml的混合物 最终浓度为1mg/ml碳水化合物(推荐分子生物学等级)

致谢

该方案的开发由授予J.T.B.的资助。 来自加拿大健康研究所。

参考文献

  1. Brimacombe,C.A.,Stevens,A.,Jun,D.,Mercer,R.,Lang,A.S.and Beatty,J.T。(2013)。 耻垢红细菌基因荚膜多糖受体的群体感应调节 转移剂(RcGTA)。 Mol Microbiol 87(4):802-817。 
  2. http://openwetware.org/images/9/9e/GLASSWARE_CLEANING_PROCEDURES.pdf
  3. Raetz,C.R。和Whitfield,C。(2002)。 脂多糖内毒素 Annu Rev Biochem 71:635- 700。
  4. Whitfield,C。(2006)。 大肠杆菌中荚膜多糖的生物合成和装配。 Annu Rev Biochem 75:39-68。
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Copyright: © 2013 The Authors; exclusive licensee Bio-protocol LLC.
引用:Brimacombe, C. A. and Beatty, J. T. (2013). Surface Polysaccharide Extraction and Quantification. Bio-protocol 3(20): e934. DOI: 10.21769/BioProtoc.934.
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