Isolation of Golgi Membranes from Tissues and Cells by Differential and Density Gradient Centrifugation
Open Access
- 1 April 2001
- journal article
- unit
- Published by Wiley in Current Protocols in Cell Biology
- Vol. 10 (1), 3.9.1-3.9.24
- https://doi.org/10.1002/0471143030.cb0309s10
Abstract
This unit describes the isolation of Golgi membranes on a preparative basis. Methods are provided for rapid isolation of dextran-treated Golgi stacks from rat liver using a sucrose density barrier, and Golgi isolation by floatation from a light mitochondrial fraction, along with an alternate procedure using a self-generated iodixinol gradient. If the Golgi tends to vesiculate during homogenization (commonly the case with cultured cells), a primary requirement is to separate these vesicles from other microsomal compartments, so a procedure is described for a discontinuous gradient of sucrose for cultured cells, and an alternate protocol describes a continuous iodixinol gradient that may provide greater resolution. A self-generated iodixinol gradient is described to prepare Golgi membranes from a microsomal fraction of rat hepatocytes, and a standard Golgi enzyme marker assay is given in a support protocol.Keywords
This publication has 24 references indexed in Scilit:
- The Transmembrane Domain of a Carboxyl-terminal Anchored Protein Determines Localization to the Endoplasmic ReticulumJournal of Biological Chemistry, 1997
- Regulated export of a secretory protein from the ER of the hepatocyte: a specific binding site retaining C-reactive protein within the ER is downregulated during the acute phase response.The Journal of cell biology, 1992
- Distribution of glycosyltransferases among Golgi apparatus subfractions from liver and hepatomas of the ratBiochimica et Biophysica Acta (BBA) - General Subjects, 1991
- Sec15 protein, an essential component of the exocytotic apparatus, is associated with the plasma membrane and with a soluble 19.5S particle.The Journal of cell biology, 1991
- Rapid analytical and preparative isolation of functional endosomes by free flow electrophoresisThe Journal of cell biology, 1987
- Reconstitution of the transport of protein between successive compartments of the golgi measured by the coupled incorporation of N-acetylglucosamineCell, 1984
- ANALYTICAL STUDY OF MICROSOMES AND ISOLATED SUBCELLULAR MEMBRANES FROM RAT LIVERThe Journal of cell biology, 1974
- GOLGI FRACTIONS PREPARED FROM RAT LIVER HOMOGENATESThe Journal of cell biology, 1973
- Preparation and characterization of golgi membranes from rat liverBiochimica et Biophysica Acta (BBA) - Biomembranes, 1970
- ISOLATION AND CHARACTERIZATION OF GOLGI MEMBRANES FROM BOVINE LIVERThe Journal of cell biology, 1969