A spin-column procedure for estrogen receptor equilibrium and competition binding analysis

摘要

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