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This collection of self-contained, "hands-on" descriptions of established laboratory protocols concentrates on receptor-mediated cell signaling, with particular attention devoted to those receptors that are part of the G-protein-linked superfamily. It offers those already working in the field as well as those in related areas the opportunity to expand their range of experimental techniques and to carry out successful investigations without needing to refer to any other methodological source.
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CHAPTER1 The Problems and Pitfalls of Radioligand Binding Mary Keen 1. Introduction Radioligand binding is an extremely versatile technique that can be applied to a wide range of receptors in a variety of preparations, including purified and solubilized receptors, membrane preparations, whole cells, tissue slices, and even whole animals. The basic method is very easy to perform. It can even be automated, and the throughput of samples that can be achieved is very high. The data obtained are typically extremely “tight” and reproducible, allowing receptor number, ligand affinity, the existence of receptor subtypes, and allosteric interactions between binding sites and/or receptors and effector molecules to be determined with great precision and subtlety. Perhaps it is the very ease of radioligand binding that presents the problem. It is extremely simple to produce data, feed the data into a computer, and generate numbers. The question of whether these numbers really mean what one hopes they mean is often overlooked. This chapter provides a brief overview of the potential problems and artifacts that may occur in radioligand binding experiments. A much more in-depth treatment of the subject can be found in ref. 1. 2. Materials and Methods 2.1. Basic Method The basic outline of all radioligand binding assays is very similar. E&ted