Measurement of Intracellular cAMP Levels Using the Cyclic Nucleotide XP Enzymatic Immunoassay Kit in Bacteria
使用环核苷酸XP酶免疫分析试剂盒测量细菌的胞内cAMP含量
发布: 2016年04月20日第6卷第8期 DOI: 10.21769/BioProtoc.1792 浏览次数: 13378
评审: Valentine V TrotterDaan C. SwartsAksiniya Asenova
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参见作者原研究论文
The authors used this protocol in:
Jun 2015
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Abstract
Cyclic AMP (cAMP) is a ubiquitous secondary signaling molecule, commonly associated with many bacterial processes, including the regulation of virulence factors, such as biofilms, pellicles and motility (Wolfgang, 2003). The quantity of available cAMP is controlled by the interplay between the synthesis of adenosine triphosphate (ATP) to cAMP by adenylyl cyclases, and the degradation of cAMP by phosphodiesterase (McDonough et al., 2012). Adequate quantification of cAMP levels within a bacterial cell is an important step in identifying the impact that secondary signaling molecules play on the regulatory pathway within the cell. The principle of this method is to measure total cAMP levels within a bacterial cell, using crude bacterial whole cell lysate. The Cyclic AMP XPTM Assay kit used in this protocol was originally designed to be used for determining the level of cAMP in eukaryotic cells, however, the antibodies used in coating the wells will react with cAMP from any species and thus can be used for determining levels in bacterial cells. The measurement of cAMP in prokaryotic cells described here is a simple and cost effective method of producing quantifiable results.
Materials and Reagents
- 1 ml cuvettes (SARSTEDT AG & Co., catalog number: 67.742 )
- 15 ml tubes (Falcon Tube) (DKSH, catalog number: LP021015 )
- 50 ml tubes (Falcon Tubes) (DKSH, catalog number: LP21050 )
- 96-well microtitre tray (tissue culture plate) (DKSH, catalog number: LP031096 )
- Aluminum foil
- Bacterial cell culture
- Mueller Hinton (MH) broth (prepared as per manufacturer’s instructions) (Oxoid Australia, catalog number: CM0405 )
- MH agar media (25 ml/Petri dish) (prepared as per manufacturer’s instructions) (Oxoid Australia, catalog number: CM0337 )
- Cyclic AMP XPTM Assay kit (Cell Signaling Technology, catalog number: 4339 )
- Protein assay DCTM reagents A & B (Bio-Rad Laboratories, catalog number: 500-121 )
- Protein assay bovine serum albumin (BSA) standards (0, 0.125, 0.25, 0.5, 1, 2.5, 5 mg/ml of protein)
- Milli Q water
- Sodium Chloride (NaCl) (Chem Supply, catalog number: SA046-5 kg )
- Potassium Chloride (KCl) (Thermo Fisher Scientific, Ajax Finechem, catalog number: A383-500 g )
- Sodium Phosphate (Na2HPO4) (Merck Millipore Corporation, catalog numer: 7558-79-4 )
- Potassium Phosphate (KH2PO4) (Merck Millipore Corporation, catalog number: 7778-77-0 )
- Phosphate buffered saline (PBS) (see Recipes)
Equipment
- VIS spectrophotometer able to read OD600 nm [e.g., DU®640 (Beckman Coulter)]
- ELISA plate reader able to read absorption at 450 nm [e.g., Multiskan EX original (Adelab Scientific)]
- French pressure cell able to operate at 30,000 psi or cell disruptor [e.g., One Shot Head from the TS series bench top disruptor (Constant Systems)]
- Ice bucket
- Sterile inoculating loop
- Shaking incubator (37 °C, at 200 rpm)
- Centrifuge (able to hold 50 ml tubes, spin at 8,000 x g and hold a pre-cooled temperature of 4 °C) [e.g. Benchtop Centrifuge Hermle Z383K (DKSH)]
- Horizontal orbital shaker (at ~30 rpm) [e.g., OM5 (Ratek, Adelab Scientific)]
Software
- Software for use with the ELISA plate reader that can export raw OD600 nm values (e.g. Microsoft Excel)
- GraphPad prism (GraphPad Software Inc)
Procedure
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文章信息
版权信息
© 2016 The Authors; exclusive licensee Bio-protocol LLC.
如何引用
Giles, S. K., Stroeher, U. H. and Brown, M. H. (2016). Measurement of Intracellular cAMP Levels Using the Cyclic Nucleotide XP Enzymatic Immunoassay Kit in Bacteria. Bio-protocol 6(8): e1792. DOI: 10.21769/BioProtoc.1792.
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分类
微生物学 > 微生物信号传导 > 第二信使
微生物学 > 微生物生物化学 > 其它化合物
生物化学 > 其它化合物 > 环磷酸腺苷（cAMP）
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