Biochemical fractionation and characterization of proteins from Golgi-enriched membranes

Fractions enriched in Golgi membranes were prepared from rat liver by sucrose gradient ultracentrifugation. These enriched membranes were further subfractionated on the basis of their solubilities in EGTA, 150 mM sodium carbonate, pH 11.5, sodium deoxycholate, Triton X-100, or sodium dodecyl sulfate. This led to isolation of peripheral, luminal, and integral membrane proteins of the Golgi-enriched membranes. Luminal and membrane proteins were further purified by wheat germ agglutinin and concanavalin A lectin affinity chromatographies. Some proteins from these lectin columns were resolved by preparative gel electrophoresis and microsequenced. Subsequently, antibodies were produced for two proteins by immunization of either mice or rabbits. Immunofluorescence microscopy suggests that these proteins are confined to Golgi apparatus-like structures. The protocol described is well suited for the study of organelle structure and function.