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

Sarah K Giles; Uwe H Stroeher; Melissa H Brown

doi:10.21769/BioProtoc.1792

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Peer-reviewed

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

Sarah K Giles

Uwe H Stroeher

Melissa H Brown email

发布: 2016年04月20日第6卷第8期 DOI: 10.21769/BioProtoc.1792 浏览次数: 12193

评审: Valentine V TrotterDaan C. SwartsAksiniya Asenova

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Cover of BMC Microbiology, featuring study using the protocol.

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 XP^TM 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 XP^TM Assay kit (Cell Signaling Technology, catalog number: 4339 )
Protein assay DC^TM 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

English

中文翻译

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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.