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Carrying on the high standards of the much-acclaimed first edition, highly experienced investigators have extensively updated the first edition with many of the new approaches that have been transforming the field. Included in this new edition are readily reproducible immunoassays, fluorescence-based assays, high-throughput methods, protein modification assays, lipid second messenger assays, and chromatin immunoprecipitation techniques. Wherever possible, protocols for the assay of general classes of signal transduction components have been identified so they can be adapted to the assay of any member of that class.
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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
From. Methods in Molecular Biology, Vol. 41’ Signal Transduction by. D. A. Kendall and S. J. HIII Copyright Q 1995 Humana Press
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Protocols Inc , Totowa,
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1. Incubate the radioligand with the receptor preparation, In parallel preparations, incubate the radioligand with the receptor preparationin the presence of an unlabeled ligand to define nonspecific binding or to investigate the binding characteristics of that unlabeled ligand (see Section 2.3.). 2. Separate bound ligand from free ligand. 3. Quantify the amount of radioligand bound, using liquid scintillation or y counting, depending on the radioisotope used. In tissue slices, autoradiog-
raphy may be used. 4. Analyze the data.
Several different types of binding assay can be performed using this basic technique. By investigating the amount of radioligand bound at various times after its addition, the association kinetics of the radioligand can be investigated. Similarly, dissociation can be investigated by allowing the radioligand and receptor to come to equilibrium, and then measuring binding at various times following infinite dilution of the sample or, more practically, following the addition of a saturating concentration of unlabeled ligand, so that the probability of radioligand binding to the receptors is reduced to close to zero. The affinity of the radioligand for the receptor and the total number of receptor sites (B-) can be determined by investigating the equilibrium binding of a range of concentrations of the radioligand. The binding of unlabeled drugs to a receptor can be measured by their ability to inhibit the specific binding of the radioligand either in equilibrium “competition” binding assays or in time-course experiments (1,2). The data from binding assays are best analyzed using computerized “curve-fitti