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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