Chapter title |
Spectrophotometric and Mass Spectroscopic Methods for the Quantification and Kinetic Evaluation of In Vitro c-di-GMP Synthesis
|
---|---|
Chapter number | 7 |
Book title |
c-di-GMP Signaling
|
Published in |
Methods in molecular biology, January 2017
|
DOI | 10.1007/978-1-4939-7240-1_7 |
Pubmed ID | |
Book ISBNs |
978-1-4939-7239-5, 978-1-4939-7240-1
|
Authors |
Geoffrey B. Severin, Christopher M. Waters |
Abstract |
The expression and activity of diguanylate cyclase (DGC) and phosphodiesterase (PDE) enzymes are responsible for modulating and maintaining the intracellular concentration of the bacterial second messenger cyclic diguanosine-monophosphate (c-di-GMP). Here, we describe an in vitro method for the spectrophotometric detection and quantification of DGC catalyzed c-di-GMP synthesis through adaptation of the EnzChek(®) Pyrophosphate Assay Kit. We also outline a method for the quantification of c-di-GMP produced in this in vitro reaction using Ultra-Performance Liquid Chromatography tandem Mass Spectrometry (UPLC-MS/MS). These methods can be leveraged for a number of experimental applications including the evaluation of enzyme activity for the in vitro synthesis of c-di-GMP, examination of how molecular signals impact these activities, identifying the catalytic properties of hybrid DGC-PDE proteins, and the development of DGC inhibitors. |
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Geographical breakdown
Country | Count | As % |
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Unknown | 8 | 100% |
Demographic breakdown
Readers by professional status | Count | As % |
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Student > Ph. D. Student | 3 | 38% |
Professor | 1 | 13% |
Other | 1 | 13% |
Unknown | 3 | 38% |
Readers by discipline | Count | As % |
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Linguistics | 1 | 13% |
Biochemistry, Genetics and Molecular Biology | 1 | 13% |
Unknown | 3 | 38% |