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Biochemical fractionation and characterization of proteins from Golgi-enriched membranes
Ist Teil von
The Journal of biological chemistry, 1992-06, Vol.267 (17), p.12016-12021
Ort / Verlag
Bethesda, MD: American Society for Biochemistry and Molecular Biology
Erscheinungsjahr
1992
Quelle
MEDLINE
Beschreibungen/Notizen
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.