植物蛋白质组学和糖基化(二)
4. 注释
( 1 ) 每个实验均使用新鲜的 3 mol/L 甲醇- HCl 和硅烷化试剂。
( 2 ) 要仔细识别蛋白质印迹,因为 WGA 既能识别 N-糖苷的 GlcNAc,也能识别 O-GlcNAc。
( 3 ) 用于在硝酸纤维素印迹膜上封闭结合位点的溶液应避免糖蛋白污染。所以我们建议在这一步骤中使用 Tween-20 来覆盖硝酸纤维素膜。
( 4 ) 特异性对照:
a. 建议印迹表 25-1 中的蛋白质到硝酸纤维素膜(建议的正对照),获得亲和检测的正对照。
b. 凝集素结合特异性应在 0.3 mol/L 抑制性糖(表 25-1 ) 浓度下进行亲和检测验证。
( 5 ) 可用的抗 O-糖苷抗体有:抗
AGP 抗体:LM2 [ 31,32] 、JIM 4、JIM 13、JIM 15 [32]、JIM 8 [33] 、JIM 14、JIM 15
和 JIM 16 [34];抗伸展蛋白抗体:LM 1 [ 35 ] 、JIM 11、 JIM 12、JIM 20 [36] 和 JIM19 [
37] 。
( 6 ) 免疫检测的 N-糖苷特异性对照:应验证血清对连接在被测蛋白的 N-糖苷的特异性,可在免疫检测前先对印迹进行温和的高碘酸盐氧化处理,温和高碘酸盐处理会氧化糖苷,并消除糖蛋白上抗苷抗体的识别位点。剩下的信号都是蛋白骨架抗体识别的结果 [ 38 ]。
a. 经明胶饱和处理后,将蛋白质印迹膜浸泡在 100 mmol/L 含 100 mmol/L 过碘酸钠的乙酸钠缓冲液(pH 4.5 ) 中,室温下黑暗处理 1 h,30 min 后更换一次浸泡液。
b. 将蛋白质印迹膜浸泡在含 50 mmol/L 硼氢化钠的 PBS 缓冲液中,室温下处理 30 min。
c. 用 TBS 漂洗蛋白质印迹膜,用含 1% 凝胶 的 TBS 浸泡蛋白质印迹膜 15 min,进行如 25. 3. 2 节 1. 1 ) 所述的免疫检测实验。
( 7 ) 岩藻糖或者木糖的特异性对照:有些蛋白质可被用作 N-糖苷免疫检测的正对照,源自蜂毒的磷脂酶含有 α-1-3岩藻糖残基,不含 β-1-2 木糖。源自玉米 的 PHA-L ( 植物血凝素 L ) 和重组抗生物素蛋白是既含 β-1-2 木糖,也含 α-1-3 岩藻糖的糖蛋白 [ 18,40 , 41 ] 。
( 8 ) 这里介绍的方法需要 1 mg 蛋白质,较少的蛋白质使用量也适用。
( 9 ) 用肌醇做内标。
( 10 ) 由于还原性氨化反应是一种激烈的处理,可能会发生一些蛋白质修饰。为此,有时候最好酶切去除 O-糖苷。
( 11 ) 总的来说,我们实验室不使用化学处理解离糖蛋白上的N - 糖苷。
( 12 ) Nonidet P40 的作用是结合游离的 SDS。
( 13 ) 需要 60~80 mg 的可溶油菜籽蛋白作为初始材料,制备足够跑一张 2D 凝胶的糖蛋白。
( 14 ) α-甲基甘露糖是刀豆蛋白 A 的配体,它将替换固定化凝集素上的糖蛋白。
( 15 ) 在洗脱亲和色谱柱时,我们观察到一个重要的解离物质刀豆蛋白 A。这一解离物质污染了糖蛋白制备物,迫使我们必须同时跑一张分析用 2D 凝胶,另一张 2D 凝胶只上样刀豆蛋白 A。染色后,我们选择只在分析用 2D 凝胶上出现的点,弃除同时在这两张 2D 凝胶上出现的点。
( 16 ) 150 ml 生长 6 天的拟南芥 cgl 突变体细胞培养物大约相当于 10 g 植物材料。
( 17 ) 细胞壁结合蛋白的去除是纯化的第一步,因为这些蛋白不含 O-位 N-乙酰葡糖胺。如果需要就应将这一纯化步骤包括在这个方法中。
( 18 ) 自由 O-位 N-乙酰葡糖胺是 WGA 的配体,它将替换固定凝集素上的糖蛋白。
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