An airfuge centrifugation procedure for the measurement of ligand binding to membrane-associated and detergent-solubilized plasma membrane receptors.
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.
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D002498:Centrifugation / Q000379:methods*; D002642:Chick Embryo; D003902:Detergents; D005260:Female; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D051379:Mice; D010920:Placenta / Q000378:metabolism; D011247:Pregnancy; D011972:Receptor, Insulin / Q000378:metabolism; D011956:Receptors, Cell Surface / Q000032:analysis / Q000378:metabolism*; D012995:Solubility; D013917:Thrombin / Q000378:metabolism
D003902:Detergents; D011956:Receptors, Cell Surface; D011972:Receptor, Insulin; D013917:Thrombin