With contributions from leading international authorities, GPCR Essential Methods equips readers with clear and detailed protocols for both well–known and up–and–coming techniques along with hints and tips for success. All the methods have been tried and tested by leading international research labs and are presented in easy–to–follow stages along with a useful overview of each technique.
This book will be of interest to researchers in molecular biology, biochemistry, pharmacology and will be a useful resource for graduate students.
- Highlights the best and most up–to–date techniques for working on GPCRs from the vast range currently in use
- Explains clearly and precisely how to carry out selected techniques in addition to background information on the various approaches
- Chapters are written by leading international authorities in the field and cover both new and well–known, tried and tested methods for working with GPCRs
- An essential laboratory manual for researchers at all levels
1 Measurement of Ligand G–Protein–coupled Receptor Interactions (Katie Leach, Celine Valant, Patrick M. Sexton and Arthur Christopoulos).
1.2 Methods and Approaches.
2 Second Messenger Assays for G–Protein–coupled Receptors: cAMP, Ca2+, Inositol Phosphates, ERK1/2 (Karen J. Gregory, Patrick M. Sexton, Arthur Christopoulos and Caroline A. Hick).
2.2 Methods and Approaches.
3 Use of the [35S]GTP S Binding Assay to Determine Ligand Efficacy at G–protein–coupled Receptors (Elodie Kara and Philip G. Strange).
3.2 Methods and Approaches.
4 Quantitative Imaging of Receptor Trafficking (Andy R. James, Takeo Awaji, F. Anne Stephenson and Nicholas A. Hartell).
4.2 Methods and Approaches.
5 Production of Recombinant G–Protein–coupled Receptor in Yeast for Structural and Functional Analysis (Richard A.J. Darby, Mohammed Jamshad, Ljuban Grgic, William J. Holmes and Roslyn M. Bill).
5.2 Methods and Approaches.
6 Monitoring GPCR Protein Complexes Using Bioluminescence Resonance Energy Transfer (Werner C. Jaeger, Kevin D.G. Pfleger and Karin A. Eidne).
6.2 Methods and Approaches.
7 Using Intramolecular Fluorescence Resonance Energy Transfer to Study Receptor Conformation (Cornelius Krasel and Carsten Hoffmann).
7.2 Methods and Approaches.
8 A Disulfide Cross–linking Strategy Useful for Studying Ligand–induced Structural Changes in GPCRs (Jian Hua Li, Stuart D.C. Ward, Sung–Jun Han, Fadi F. Hamdan and Jürgen Wess).
8.2 Methods and Approaches.
8.3 General Considerations, Caveats and Troubleshooting.
9 Use of Fluorescence Correlation Spectroscopy to Study the Diffusion of G–protein–coupled Receptors (Stephen J. Briddon, Jonathan A. Hern and Stephen J. Hill).
9.2 Methods and Approaches.
10 Identification and Proteomic Analysis of GPCR Phosphorylation (Kok Choi Kong, Sharad C. Mistry and Andrew B. Tobin).
11 Measurement and Visualization of G–Protein–coupled Receptor Trafficking by Enzyme–linked Immunosorbent Assay and Immunofluorescence (Stuart J. Mundell, Shaista P. Nisar and Eamonn Kelly).
11.2 Methods and Approaches.
12 Substituted Cysteine Accessibility Method (SCAM) (George Liapakis and Jonathan A. Javitch).
12.2 Methods and Approaches.
13 Homology Modelling of G–Protein–coupled Receptors (John Simms).
13.2 Methods and Approaches.
13.4 Automated Methods for Generating Models of GPCRs.
Appendix: Site–directed Mutagenesis and Chimeras (Alex Conner, Mark Wheatley and David Poyner).
A.2 Why Mutagenesis?