An airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors

Abstract

A method is described in which high-speed centrifugation of membranes through an oil phase is used to separate membrane-bound and detergent-solubilized polypeptide receptor-iodinated ligand complexes from unbound ligands. Three centrifuges, the Brinkmann Eppendorf (5412), the Beckman Microfuge B and the Beckman Airfuge were evaluated for this capability. Under the conditions described, the Beckman Airfuge surpassed the others in recovering previously 125I- and 32P-labelled cell membranes. The Airfuge method was compared with the more classically employed membrane filtration method to measure specific [125I]insulin and [125I]thrombin binding to human placental membranes and an enriched plasma membrane fraction from mouse embryo fibroblasts, respectively, are found to be 4 to 6 times more sensitive. For example, specific binding of ligand to its receptor was demonstrated with 5 micrograms of protein. With slight modifications, the polyethyleneglycol 6000 method of precipitating 125I-labelled ligand-soluble receptor complexes can be adapted to the Airfuge sedimentation through oil procedure.