Characterization of cytoplasmically oriented Golgi proteins with a monoclonal antibody.
BALB/c mice were repeatedly immunized with a galactosyl transferase-rich microsomal fraction of rat myeloma cells. Spleen cells were subsequently fused with Sp2/0 mouse myeloma cells, the resulting hybridomas were cloned, and their secreted Ig was screened for reactivity with antigens belonging to the Golgi complex. One such monoclonal antibody, 6F4C5, gave especially intense immunofluorescent staining of the Golgi area of myeloma cells and fibroblasts. It recognized two proteins bands on immunoblots of gel-fractionated cell lysates: a major one with an estimated Mr of 54,000 and a minor one at 86,000. Both proteins were concentrated in microsomal fractions isolated at low ionic strength. They were hydrophilic judging from partitioning of a Triton X-114 cell lysate. Both were cytoplasmically oriented as demonstrated by protease and high KCl treatments of postmitochondrial supernatants and microsomal fractions. Neither was retained by columns of insolubilized wheat germ agglutinin or concanavalin A, which suggests that they are not glycoproteins. Their more detailed location in the Golgi complex was studied by immunoelectron microscopy, using a saponin permeabilization procedure and peroxidase-conjugated reagents. The observed staining was restricted to two or three cisternae in the medial part of the stack. Nevertheless, differential centrifugation experiments indicated that the two antigens may be recovered in distinct subcellular fractions: this may be related to the unexpected observation that rather low salt concentrations strip the antigens from microsomal fraction.
D000818:Animals; D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D002460:Cell Line; D005455:Fluorescent Antibody Technique; D005700:Galactosyltransferases / Q000032:analysis; D006056:Golgi Apparatus / Q000648:ultrastructure*; D008854:Microscopy, Electron; D008861:Microsomes / Q000648:ultrastructure; D009363:Neoplasm Proteins / Q000032:analysis*; D010954:Plasmacytoma / Q000648:ultrastructure*; D051381:Rats; D011975:Receptors, Mitogen / Q000032:analysis
D000911:Antibodies, Monoclonal; D000936:Antigen-Antibody Complex; D009363:Neoplasm Proteins; D011975:Receptors, Mitogen; D005700:Galactosyltransferases