PRACTICAL INSTRUCTIONS FOR RADIOACTIVELY LABELED LIGAND-RECEPTOR BINDING-STUDIES

This paper represents a practical guide to ligand receptor binding stu dies with special emphasis on the detailed characterization of radiola beled ligands prior to receptor binding experiments. The effects due t o labeled ligand inactivated by the labeling procedure are included. T he inactive ligand may contribute as much as the active ligand to nons pecific binding. A method for measuring the fraction of active ligand in the initial sample and for determining the specific radioactivity a nd bindability is presented in detail. A straightforward procedure for extracting the concentration of bound and free active ligand from the measured crude binding data is then presented. The dissociation const ant and the number of receptors are obtained by fitting the binding eq uation to the free and bound ligand concentrations. As a practical exa mple, interleukin-8 binding to human neutrophils was used. The experim ents are discussed with respect to possible errors, and specific condi tions (concentrations of ligand and receptors) necessary for accurate determination of the respective binding parameters are given. (C) 1994 Academic Press, Inc.