Immuno-isolation of a plasma membrane fraction from the Fao cell.
A plasma membrane was immuno-isolated from a post-nuclear supernatant of a cultured rat hepatocyte, the Fao cell, using a cellulose immuno-adsorbent and antibodies raised against a variety of endogenous antigens of hepatocytes: 5'-nucleotidase, a plasma membrane fraction and the whole Fao cell. The antibodies which recognize antigens on the cell surface were selected from the total serum by first binding the antiserum to suspension cells. Alternatively, the plasma membrane and Fao antisera were affinity purified on a column prepared from a Triton X-114 extract of a plasma membrane fraction. The immuno-isolation was most efficient when carried out with either the plasma membrane or the Fao anti-serum. When alkaline phosphodiesterase I or 5'-nucleotidase was used as the plasma membrane marker, 40-60% of the plasma membrane of the post-nuclear supernatant was isolated representing a maximum 34-fold increase in the specific activity of the enzymes in the bound material. Using the NaB-[3H]4-labelled glycoproteins of the plasma membrane or the IgG bound to the plasma membrane as alternative markers, an 80% isolate of the plasma membrane of the post-nuclear supernatant was achieved, resulting in an estimated 40-fold purification. The non-specific binding was low despite the use of a post-nuclear supernatant as the input fraction. The characterization of the bound materials suggested that the whole plasma membrane was immuno-isolated and not a particular domain.
D000818:Animals; D000906:Antibodies; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface / Q000032:analysis; D002458:Cell Fractionation / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000648:ultrastructure*; D005692:Galactose Oxidase / Q000032:analysis; D007074:Immunoglobulin G; D008114:Liver Neoplasms, Experimental / Q000648:ultrastructure*; D008854:Microscopy, Electron; D051381:Rats
D000906:Antibodies; D000936:Antigen-Antibody Complex; D000954:Antigens, Surface; D007074:Immunoglobulin G; D005692:Galactose Oxidase