Abstract

Typical equilibrium binding experiments cannot be quantitatively analyzed on the basis of classical mathematical equations when the receptor concentration is so high that a significant fraction of the added radioligand concentration is in the bound form. In this paper, the appropriate equations are derived and used in a commercial graphics package to estimate the binding parameters, by applying nonlinear regression to pseudo-experimental data. The analysis of saturation and homologous displacement curves obtained with high receptor concentrations reveals that the empirical determination of nonspecific binding by addition of an excess of unlabeled ligand is incorrect.