By David Poyner, Mark Wheatley
G-Protein Coupled Receptors (GPCRs) aren't in simple terms the most important protein family members within the human genome yet also are the only largest aim for healing brokers. examine into GPCRs is as a result turning out to be at a quick velocity and the variety of strategies that may be utilized to GPCRs is tremendous and maintains to develop. This e-book presents a useful bench-side consultant into the easiest and most modern innovations for present and destiny study on GPCRs.
With contributions from top overseas authorities, this book equips readers with transparent and distinctive protocols for either famous and up-and-coming suggestions besides tricks and tips for achievement. the entire tools were attempted and demonstrated through prime foreign learn labs and are offered in easy-to-follow levels in addition to an invaluable review of every strategy.
This ebook is a necessary source for all researchers in molecular biology, biochemistry, pharmacology and for graduate students.Content:
Chapter 1 size of Ligand–G Protein?Coupled Receptor Interactions (pages 1–29): Katie Leach, Celine Valant, Patrick M. Sexton and Arthur Christopoulos
Chapter 2 moment Messenger Assays for G Protein?Coupled Receptors: cAMP, Ca2+, Inositol Phosphates, ERK1/2 (pages 31–52): Karen J. Gregory, Patrick M. Sexton, Arthur Christopoulos and Caroline A. Hick
Chapter three Use of the [35S]GTP?S Binding Assay to figure out Ligand Efficacy at G Protein?Coupled Receptors (pages 53–68): Elodie Kara and Philip G. Strange
Chapter four Quantitative Imaging of Receptor Trafficking (pages 69–83): Andy R. James, Takeo Awaji, F. Anne Stephenson and Nicholas A. Hartell
Chapter five construction of Recombinant G Protein?Coupled Receptor in Yeast for Structural and practical research (pages 85–110): Richard A. J. Darby, Mohammed Jamshad, Ljuban Grgic, William J. Holmes and Roslyn M. Bill
Chapter 6 tracking GPCR–Protein Complexes utilizing Bioluminescence Resonance power move (pages 111–132): Werner C. Jaeger, Kevin D. G. Pfleger and Karin A. Eidne
Chapter 7 utilizing Intramolecular Fluorescence Resonance strength move to review Receptor Conformation (pages 133–146): Cornelius Krasel and Carsten Hoffmann
Chapter eight A Disulfide Cross?linking process valuable for learning Ligand?induced Structural alterations in GPCRs (pages 147–167): Jian Hua Li, Stuart D. C. Ward, Sung?Jun Han, Fadi F. Hamdan and Jurgen Wess
Chapter nine Use of Fluorescence Correlation Spectroscopy to check the Diffusion of G Protein?coupled Receptors (pages 169–195): Stephen J. Briddon, Jonathan A. Hern and Stephen J. Hill
Chapter 10 identity and research of GPCR Phosphorylation (pages 197–214): Kok Choi Kong, Sharad C. Mistry and Andrew B. Tobin
Chapter eleven dimension and Visualization of G Protein?coupled Receptor Trafficking by means of Enzyme?linked Immunosorbent Assay and Immunofluorescence (pages 215–228): Stuart J. Mundell, Shaista P. Nisar and Eamonn Kelly
Chapter 12 Substituted Cysteine Accessibility procedure (SCAM) (pages 229–250): George Liapakis and Jonathan A. Javitch
Chapter thirteen Homology Modelling of G Protein?Coupled Receptors (pages 251–273): John Simms
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Extra resources for G Protein-Coupled Receptors: Essential Methods
2 Spin the cells at 2500 g for 4 min in a bench-top centrifuge and resuspend the cells in 10 ml of complete growth media without antibiotics. 3 Count the number of cells using a haemocytometer and seed the cells at 30 000–50 000 cells per well in 96-well black clear-bottom plates using 200 μl of cell suspension per well. 4 Incubate the cells in the 96-well plates for 24 h at 37 ◦ C and 5% CO2 . At the time of the assay the cells should be 90–100% confluent. 5 Make up assay buffer on the day of the experiment; warm buffer to 37 ◦ C.
Pharmacol. , 14, 48–58. One of the original publications describing concepts of competitive and noncompetitive antagonism and the use of the ‘Schild plot’. REFERENCES 29 19. M. and Christopoulos, A. (2004) Application of a kinetic model to the apparently complex behavior of negative and positive allosteric modulators of muscarinic acetylcholine receptors. J. Pharmacol. Exp. , 308, 1062–1072. 20. Lazareno, S. J. (1995) Detection, quantitation, and verification of allosteric interactions of agents with labeled and unlabeled ligands at G protein-coupled receptors: interactions of strychnine and acetylcholine at muscarinic receptors.
B Pluronic F-127 is a nonionic detergent which can assist in dispersion of the nonpolar Fluo-4 AM ester in aqueous media, thereby increasing cell loading. c Ionomycin is a calcium ionophore and can be used to record a maximum calcium response. d UTP acts on P2Y receptors endogenously expressed in most cell lines and can be used as an internal control. e When washing the cells, handle the plates carefully to avoid dislodging the cells from the bottom of the wells. 44 CH 2 SECOND MESSENGER ASSAYS FOR G PROTEIN-COUPLED RECEPTORS f It may be necessary to perform an agonist dose–response curve in the presence of antagonist to determine the most suitable agonist concentration to use.