Subramaniam V N, bin Mohd Yusoff A R, Wong S H, Lim G B, Chew M, Hong W
Laboratory of Molecular and Cell Biology of Membranes, National University of Singapore.
J Biol Chem. 1992 Jun 15;267(17):12016-21.
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.
通过蔗糖梯度超速离心从大鼠肝脏中制备富含高尔基体膜的组分。这些富集的膜根据它们在乙二醇双乙醚二胺四乙酸（EGTA）、150 mM碳酸钠（pH 11.5）、脱氧胆酸钠、曲拉通X-100或十二烷基硫酸钠中的溶解度进一步细分。这导致了高尔基体富集膜的外周、腔内和整合膜蛋白的分离。腔内和膜蛋白通过麦胚凝集素和伴刀豆球蛋白A凝集素亲和层析进一步纯化。来自这些凝集素柱的一些蛋白质通过制备性凝胶电泳分离并进行微量测序。随后，通过免疫小鼠或兔子产生了针对两种蛋白质的抗体。免疫荧光显微镜检查表明这些蛋白质局限于高尔基体样结构。所描述的方案非常适合细胞器结构和功能的研究。
