ABSTRACT
Estrogen receptors, members of the nuclear hormone receptor family, are not only able to bind their endogenous hormone, 17β-estradiol, but can also accommodate other naturally-occuring, non-steroidal molecules. Here, we describe a spin-column procedure to determine accurately equilibrium dissociation constants (Kds) and IC50 concentrations for estrogenic compounds. The human wild-type ERα was used to validate the protocol. We expressed the full-length ERα protein in an eukaryotic system to ensure all possible post-transcriptional modifications. The gel filtration-based assay revealed a temperature-dependent Kd shift for ERα. At physiological conditions (150 mM salt, 37°C) we determined the 17β-estradiol Kd for ERα to be 281 ± 13 pmol/L. Positive cooperativity was only apparent at low temperatures and diminished to zero at 37°C. In homologous competition binding experiments using 17β-estradiol, we observed fifty fold higher IC50 values than the respective Kd. This paper presents a reliable and sensitive protocol to generate saturation binding curves and heterologous competition curves to test estrogenic compounds.