This volume provides a collection of protocols for the common experimental approaches used in the in the burgeoning field of c-di-GMP-dependent signaling. The chapters, divided into eight major parts, guide readers through methods on synthesis, detection, quantitation, modulation of the levels of c-di-GMP present in cells, procedures to detect and evaluate the interaction of c-di-GMP, and up and coming approaches focusing on the inhibition of c-di-GMP signaling.Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective…mehr
This volume provides a collection of protocols for the common experimental approaches used in the in the burgeoning field of c-di-GMP-dependent signaling. The chapters, divided into eight major parts, guide readers through methods on synthesis, detection, quantitation, modulation of the levels of c-di-GMP present in cells, procedures to detect and evaluate the interaction of c-di-GMP, and up and coming approaches focusing on the inhibition of c-di-GMP signaling.Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and cutting-edge, c-di-GMP Signaling: Methods and Protocols aims to inspire researchers to try new approaches.
Discovery of the Intracellular Signaling Molecule Cyclic di-GMP.- Enzymatic Production of C-di-GMP Using a Thermophilic Diguanylate Cyclase.- Synthesis of [32P]-c-di-GMP for Diguanylate Cyclase and Phosphodiesterase Activity Determinations.- High-performance Liquid Chromatography (HPLC)-based Detection and Quantitation of Cellular c-di-GMP.- Identification and Quantification of Cyclic Di-Guanosine Monophosphate and its Linear Metabolites by Reversed-Phase LC-MS/MS.- Detection of Cyclic Dinucleotides by STING.- Spectrophotometric and Mass Spectroscopic Methods for the Quantification and Kinetic Evaluation of In Vitro c-di-GMP Synthesis.- Gauging and Visualizing c-di-GMP Levels in Pseudomonas aeruginosa using Fluorescence-based Biosensors.- Cyclic di-GMP-responsive Transcriptional Reporter Bioassays in Pseudomonas aeruginosa .- Live flow cytometry analysis of c-di-GMP levels in Single Cell Populations.- Experimental Detection and Visualization of the Extracellular Matrix in Macrocolony Biofilms.- Congo Red Stain Identifies Matrix Overproduction and is an Indirect Measurement for c-di-GMP in many Species of Bacteria.- Type IV pili-dependent Motility as a Tool to Determine the Activity of c-di-GMP Modulating Enzymes in Myxococcus xanthus.- Using Light-Activated Enzymes for Modulating Intracellular c di-GMP Levels in Bacteria.- Analysis of c-di-GMP Levels Synthesized by a Photoreceptor Protein in Response to Different Light Qualities using an in vitro Enzymatic Assay.- Probing the Role of Cyclic di-GMP Signalling Systems in Disease using Chinese Radish.- Contribution of Cyclic di-GMP in the Control of Type III and Type VI Secretion in Pseudomonas aeruginosa.- Semi-quantitative Analysis of the Red, Dry, and Rough Colony Morphology of Salmonella enterica serovar Typhimuriumand Escherichia coli using Congo Red.- Fluorescent 2-aminopurine c-di-GMP and GpG analogs as PDE Probes.- Measuring Cyclic Diguanylate (c-di-GMP)-specific Phosphodiesterase Activity using the MANT-c-di-GMP Assay.- Determining Phosphodiesterase Activity (Radioactive Assay).- Determining Diguanylate Cyclase Activity (Radioactive Assay).- Detection of c-di-GMP-responsive DNA Binding.- Use of Nonradiochemical DNAse Footprinting to Analyze c-di-GMP Modulation of DNA-binding Proteins.- Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pulldown Assay.- Probing Protein-protein Interactions with Genetically Encoded Photo-activatable Crosslinkers.- Identification of c-di-AMP-Binding Proteins Using Magnetic Beads.- Pulldown with a c-di-GMP-specific Capture Compound Coupled to Mass Spectrometry as a Powerful Tool to Identify Novel Effector Proteins.- Identification of c-di-GMP-responsive Riboswitches.- Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (Kd) and Stoichiometry of Interaction (n) of c-di-GMP Binding Proteins.- Targeting c-di-GMP Signaling, Biofilm Formation and Bacterial Motility With Small Molecules.- Discovering Selective Diguanylatecyclases Inhibitors: From PleD to Discrimination of the Active Site of cyclic-di-GMP Phosphodiesterases.- High throughput Screening for Compounds that Modulate the Cellular c-di-GMP Level in Bacteria.- Genetic Tools to Study c-di-GMP-dependent Signaling in Pseudomonas aeruginosa.
Discovery of the Intracellular Signaling Molecule Cyclic di-GMP.- Enzymatic Production of C-di-GMP Using a Thermophilic Diguanylate Cyclase.- Synthesis of [32P]-c-di-GMP for Diguanylate Cyclase and Phosphodiesterase Activity Determinations.- High-performance Liquid Chromatography (HPLC)-based Detection and Quantitation of Cellular c-di-GMP.- Identification and Quantification of Cyclic Di-Guanosine Monophosphate and its Linear Metabolites by Reversed-Phase LC-MS/MS.- Detection of Cyclic Dinucleotides by STING.- Spectrophotometric and Mass Spectroscopic Methods for the Quantification and Kinetic Evaluation of In Vitro c-di-GMP Synthesis.- Gauging and Visualizing c-di-GMP Levels in Pseudomonas aeruginosa using Fluorescence-based Biosensors.- Cyclic di-GMP-responsive Transcriptional Reporter Bioassays in Pseudomonas aeruginosa .- Live flow cytometry analysis of c-di-GMP levels in Single Cell Populations.- Experimental Detection and Visualization of the Extracellular Matrix in Macrocolony Biofilms.- Congo Red Stain Identifies Matrix Overproduction and is an Indirect Measurement for c-di-GMP in many Species of Bacteria.- Type IV pili-dependent Motility as a Tool to Determine the Activity of c-di-GMP Modulating Enzymes in Myxococcus xanthus.- Using Light-Activated Enzymes for Modulating Intracellular c di-GMP Levels in Bacteria.- Analysis of c-di-GMP Levels Synthesized by a Photoreceptor Protein in Response to Different Light Qualities using an in vitro Enzymatic Assay.- Probing the Role of Cyclic di-GMP Signalling Systems in Disease using Chinese Radish.- Contribution of Cyclic di-GMP in the Control of Type III and Type VI Secretion in Pseudomonas aeruginosa.- Semi-quantitative Analysis of the Red, Dry, and Rough Colony Morphology of Salmonella enterica serovar Typhimuriumand Escherichia coli using Congo Red.- Fluorescent 2-aminopurine c-di-GMP and GpG analogs as PDE Probes.- Measuring Cyclic Diguanylate (c-di-GMP)-specific Phosphodiesterase Activity using the MANT-c-di-GMP Assay.- Determining Phosphodiesterase Activity (Radioactive Assay).- Determining Diguanylate Cyclase Activity (Radioactive Assay).- Detection of c-di-GMP-responsive DNA Binding.- Use of Nonradiochemical DNAse Footprinting to Analyze c-di-GMP Modulation of DNA-binding Proteins.- Detection of Cyclic di-GMP Binding Proteins Utilizing a Biotinylated Cyclic di-GMP Pulldown Assay.- Probing Protein-protein Interactions with Genetically Encoded Photo-activatable Crosslinkers.- Identification of c-di-AMP-Binding Proteins Using Magnetic Beads.- Pulldown with a c-di-GMP-specific Capture Compound Coupled to Mass Spectrometry as a Powerful Tool to Identify Novel Effector Proteins.- Identification of c-di-GMP-responsive Riboswitches.- Isothermal Titration Calorimetry to Determine Apparent Dissociation Constants (Kd) and Stoichiometry of Interaction (n) of c-di-GMP Binding Proteins.- Targeting c-di-GMP Signaling, Biofilm Formation and Bacterial Motility With Small Molecules.- Discovering Selective Diguanylatecyclases Inhibitors: From PleD to Discrimination of the Active Site of cyclic-di-GMP Phosphodiesterases.- High throughput Screening for Compounds that Modulate the Cellular c-di-GMP Level in Bacteria.- Genetic Tools to Study c-di-GMP-dependent Signaling in Pseudomonas aeruginosa.
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