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植物材料
拟南芥生态型Columbia-0在22℃下在土壤上生长,短日照(9小时光照/ 15小时黑暗,170μE)和70%相对湿度。6-8周后收获绿叶(包括叶柄)并用于质膜分离。
质膜隔离(不翻译了,怕错)About 100 g of plant material was homogenized, using a knife blender, in 150 ml of 330 mM sucrose, 50 mM MOPS-KOH, pH 7.5, 5 mM EDTA, 0.2% casein hydrolysate, 0.6% polyvinylpolypyrrolidone (PVPP), 5 mM ascorbate, 5 mM DTT. PVPP, ascorbate and DTT were added immediately before use. Immediately after homogenization, PMSF was added to a final concentration of 0.5 mM together with 1.5 ml of a protease inhibitor cocktail for plant cell and tissue extracts (P9599; Sigma, St. Louis, MO, U.S.A.) in DMSO. The homogenate was filtered through a 200 µm nylon mesh and centrifuged at 10,000×g for 15 min; the supernatant was saved and centrifuged at 30,000×g for 50 min. The resulting microsomal pellet was resuspended in 6 ml of resuspension medium: 330 mM sucrose, 5 mM K-phosphate, pH 7.8, 0.1 mM EDTA, 1 mM DTT (freshly added) and 50 µl protease inhibitor cocktail. The resuspended membran