Production and characterization of antibodies specific for the imidazoline receptor protein

Abstract

Polyclonal antibodies were raised in rabbits against a 70-kDa ligand-binding protein of the imidazoline receptor purified from solubilized bovine adrenal chromaffin cell membranes by ligand affinity chromatography. The antibodies labeled a single protein (approximately 70 kDa) in Western blots of bovine adrenal chromaffin cell membranes, inhibited 40% of specific [3H]idazoxan binding to imidazoline receptors in chromaffin cell membranes, and specifically immunoprecipitated 75% of all imidazoline-binding activity of solubilized chromaffin cell membrane proteins. The antibodies specifically immunostained heterogeneous subsets of cultured bovine chromaffin cells. They stained subpopulations of chromaffin cells of rat adrenal medulla but not the cells of adrenal cortex. We conclude that the antibodies recognize with high specificity and selectivity a approximately 70-kDa binding protein associated with or representing the imidazoline receptor that is expressed in mammalian species. Highly specific antibodies against the imidazoline receptor protein will permit mapping of the distribution of imidazoline receptors in brain and periphery and also may be useful as probes in cloning genes encoding the imidazoline receptors.