Practical instructions for radioactively labeled ligand receptor binding studies

This paper represents a practical guide to ligand receptor binding studies with special emphasis on the detailed characterization of radiolabeled ligands prior to receptor binding experiments. The effects due to labeled ligand inactivated by the labeling procedure are included. The inactive ligand may contribute as much as the active ligand to nonspecific binding. A method for measuring the fraction of active ligand in the initial sample and for determining the specific radioactivity and 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 constant and the number of receptors are obtained by fitting the binding equation to the free and bound ligand concentrations. As a practical example, interleukin-8 binding to human neutrophils was used. The experiments are discussed with respect to possible errors, and specific conditions (concentrations of ligand and receptors) necessary for accurate determination of the respective binding parameters are given.