Extraction and Quantification of Cyclic Di-GMP from Pseudomonas aeruginosa
从绿脓杆菌中提取并量化环鸟苷二磷酸
发布: 2013年07月20日第3卷第14期 DOI: 10.21769/BioProtoc.828 浏览次数: 14514
评审: Fanglian He
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参见作者原研究论文
The authors used this protocol in:
Jun 2012
Abstract
Cyclic di-GMP (c-di-GMP) has emerged as an important intracellular signaling molecule, controlling the transitions between planktonic (free-living) and sessile lifestyles, biofilm formation, and virulence in a wide variety of microorganisms. The following protocol describes the extraction and quantification of c-di-GMP from Pseudomonas aeruginosa samples. We have made every effort to keep the protocol as general as possible to enable the procedure to be applicable for the analysis of c-di-GMP levels in various bacterial species. However, some modifications may be required for the analysis of c-di-GMP levels in other bacterial species.
Keywords: HPLC (高效液相色谱法)
Biofilm (生物膜)
Standard curve (标准曲线)
Calibration (校准)
Materials and Reagents
- Bacterial culture
- Ethanol (95-100%)
- Tris base
- EDTA
- NaCl
- KCl
- Na2HPO4.7H2O
- KH2PO4 (pH 7.2)
- 0.5 M Ethylenediaminetetraacetic acid (EDTA) disodium salt solution (pH 8.0)
- Bis-(3'-5')-cyclic diguanylic monophosphate (c-di-GMP) (Biolog)
- Ammonium acetate (MS grade)
- Methanol (HPLC grade)
- Nanopure water (18 Ohm)
- Protein determination reagents
- Phosphate-buffered saline (PBS) (see Recipes)
- HPLC Solvent A (see Recipes)
- HPLC Solvent B (see Recipes)
- TE buffer (see Recipes)
Equipment
- Syringes (1 ml)
- Syringe filters (2 μm) (Upchurch Scientific, catalog number: B-100 )
- Microfuge tubes (unpolished, to reduce static)
- Spectrophotometer
- Refrigerated microcentrifuge
- Vacuum concentrator/Centrifugal evaporator (e.g. SpeedVac)
- Reverse-phase C18 Targa column (2.1 x 40 mm, 5 μm) (The Nest Group, catalog number: TR-0421-C185 )
- Heat block or water bath
- High performance liquid chromatography (HPLC) system and software for HPLC peak analysis (e.g. Agilent 1100 HPLC)
- Sonicator
- Homogenizer
Software
- ChemStation for LC (Agilent Technologies)
Procedure
文章信息
版权信息
© 2013 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Roy, A. B., Petrova, O. E. and Sauer, K. (2013). Extraction and Quantification of Cyclic Di-GMP from Pseudomonas aeruginosa. Bio-protocol 3(14): e828. DOI: 10.21769/BioProtoc.828.
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分类
微生物学 > 微生物生物化学 > 其它化合物
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