Titlebook: G Protein-Coupled Receptor Screening Assays; Methods and Protocol Sofia Aires M. Martins,Duarte Miguel F. Prazeres Book 2021Latest edition

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Imaging of Genetically Encoded FRET-Based Biosensors to Detect GPCR Activity,nable dynamic measurements. Moreover, FRET biosensors are ideally suited for the analysis of single living cells. The FRET biosensors described in this manuscript are entirely genetically encoded by plasmids. Here, protocols for employing FRET-based biosensors to detect G protein activity upon GPCR

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cAMP Biosensor Assay Using BacMam Expression System: Studying the Downstream Signaling of LH/hCG Rehe biological activity of various GPCR-specific agents, including protein hormones. cAMP biosensors based on detection of Förster-type resonance energy transfer (FRET) offer unique advantages including the ratiometric nature of measurement, adjustable affinity toward detected molecule, capability of

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FLIPR Calcium Mobilization Assays in GPCR Drug Discovery,fluorescence-based method (FLIPR Calcium 4 Assay) developed by Molecular Devices for a FlexStation and routinely used in our laboratory for detecting intracellular calcium changes. The assay is designed to study calcium mobilization induced by majority of GPCRs and calcium channels and allows for si

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Split-Tobacco Etch Virus (Split-TEV) Method in G Protein-Coupled Receptor Interacting Proteins,(TEV) protease in two fragments, where each fragment is fused to the candidate proteins predicted to interact. If there is indeed an interaction between both proteins, TEV protease reconstitutes its proteolytic activity and this activity is used to induce the expression of some reporter genes. Howev

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Gradient Tracking by Yeast GPCRs in a Microfluidics Chamber,n provide precise control of the time, dose, and orientation of a stimulus, while simultaneously capturing quantitative single-cell data. The approach is particularly powerful when combined with the genetically tractable yeast model organism. The GPCR pathway in yeast is structurally conserved and f

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Monitoring Intracellular Calcium in Response to GPCR Activation: Comparison Between Microtiter Platr speeds and lower costs, while at the same time mimicking the local chemical concentrations and force fields of the cellular in vivo environment. In this chapter we introduce a microfluidic structure with hydrodynamic cell traps and a culture volume in the nanoliter range (50 nL), to quantitatively

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