title: Immunopurification of Golgi vesicles by magnetic sorting.
abstract: We have designed a method that permits to isolate highly purified Golgi vesicles deprived of endoplasmic reticulum (ER), main contaminant of Golgi fractions. To this end, we prepared a rabbit polyclonal antibody against the cytosolic N-terminal oligopeptide of the enzyme heparan glucosaminyl N-deacetylase/N-sulphotransferase (HSST), a specific marker for Golgi apparatus. The Golgi localization of HSST was confirmed by indirect immunofluorescence microscopy. The antibody binding to Golgi vesicles was demonstrated by immunoelectronmicroscopy and allowed the immunopurification by magnetic sorting. Golgi vesicles subjected to purification by magnetic sorting showed the presence of HSST and p28, which is an integral membrane protein on the cis-Golgi also used as a specific Golgi marker. The purified material was devoid of calreticulin, a specific ER marker. This purification method will allow to improve studies requiring highly purified Golgi membranes such as identification of specific receptors and the electrophysiological characterization of Golgi membrane ion channels, which have been jeopardized up to now by ER membrane contamination.
authors.affiliation: Departamento de Biología, Facultad de Ciencias, Universidad de Chile, Las Palmeras 3425, Casilla 653, Santiago, Chile. cmura@uchile.cl
authors.forename: Casilda V
authors.initials: CV
authors.lastname: Mura
authors.forename: María Inés
authors.initials: MI
authors.lastname: Becker
authors.forename: Ariel
authors.initials: A
authors.lastname: Orellana
authors.forename: Daniel
authors.initials: D
authors.lastname: Wolff
mesh_terms: D000581:Amidohydrolases; D000818:Animals; D006056:Golgi Apparatus; D007150:Immunohistochemistry; D018189:Immunomagnetic Separation / Q000379:methods; D008856:Microscopy, Fluorescence; D011817:Rabbits; D015238:Sulfotransferases
chemical_list: C113066:NDST2 protein, human; D015238:Sulfotransferases; D000581:Amidohydrolases