Immunopurification of Golgi vesicles by magnetic sorting

Casilda V. Mura, María Inés Becker, Ariel Orellana, Daniel Wolff

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59 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)263-271
Number of pages9
JournalJournal of Immunological Methods
Volume260
Issue number1-2
DOIs
Publication statusPublished - 1 Feb 2002

Keywords

  • Golgi vesicles
  • Heparan glucosaminyl N-deacetylase/N-sulphotransferase
  • Immunopurification
  • Magnetic sorting

ASJC Scopus subject areas

  • Immunology and Allergy
  • Immunology

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