Isolation of Golgi membranes from tissues and cells by differential and density gradient centrifugation.
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.
Graham
J M
JM
Liverpool John Moores University, Liverpool, United Kingdom.
D000818:Animals; D015415:Biomarkers; D016466:CHO Cells / Q000648:ultrastructure; D002458:Cell Fractionation / Q000379:methods*; D002498:Centrifugation / Q000379:methods*; D002499:Centrifugation, Density Gradient / Q000379:methods; D006224:Cricetinae; D003412:Cricetulus; D003911:Dextrans; D005700:Galactosyltransferases / Q000032:analysis; D006056:Golgi Apparatus / Q000201:enzymology; D022781:Hepatocytes / Q000737:chemistry / Q000648:ultrastructure*; D051379:Mice; D009237:N-Acetyllactosamine Synthase / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013395:Sucrose; D014283:Triiodobenzoic Acids; D000520:alpha-Glucosidases / Q000032:analysis
D015415:Biomarkers; D003911:Dextrans; D014283:Triiodobenzoic Acids; D013395:Sucrose; D005700:Galactosyltransferases; D009237:N-Acetyllactosamine Synthase; C042059:glucosidase I; D000520:alpha-Glucosidases; C044834:iodixanol