Golgi: Methods for Preparation

Abstract

The study of Golgi structure and function has been greatly facilitated by the purification of this membrane organelle from cellular homogenates. Purified Golgi membranes have been used in a variety of cell‐free assays to investigate sugar modifications, vesicle transport, Golgi structure formation and Golgi–cytoskeleton interactions. Golgi membranes can be purified from cells and tissues using a number of different methods, with liver as a preferred source. Highly purified Golgi stacks can be obtained after two sequential density gradient centrifugations of rat liver homogenate. The relative purity of the prepared Golgi stacks is assessed by measuring the increase in activity of a Golgi enzyme, β‐1,4‐galactosyltransferase (GalT), over that of the total liver homogenate. A typical preparation can yield milligrams of Golgi membranes that are purified 80‐ to 100‐fold over the homogenate and 60–70% in stacks, which provides abundant material for both structural and functional studies of this organelle. Key Concepts: The Golgi apparatus is an essential membrane‐bound organelle in the centre of the secretory pathway in almost all eukaryotic cells. The primary function of the Golgi is to modify and package proteins and lipids into transport carriers and send them to the proper locations. Golgi can be separated from other membranous organelles by density gradient centrifugation. Glycosyl transferases are used as marker enzymes to monitor the yield and purity of the purified Golgi apparatus during Golgi preparation. Purified Golgi membranes can be used in a variety of cell‐free assays to investigate sugar modifications, vesicle transport, Golgi structure formation and Golgi–cytoskeleton interactions.