4. 注釋
( 1 ) 每個實驗均使用新鮮的 3 mol/L 甲醇- HCl 和硅烷化試劑。
( 2 ) 要仔細識別蛋白質印跡，因為 WGA 既能識別 N-糖苷的 GlcNAc，也能識別 O-GlcNAc。
( 3 ) 用于在硝酸纖維素印跡膜上封閉結合位點的溶液應避免糖蛋白污染。所以我們建議在這一步驟中使用 Tween-20 來覆蓋硝酸纖維素膜。
( 4 ) 特異性對照：
a. 建議印跡表 25-1 中的蛋白質到硝酸纖維素膜（建議的正對照），獲得親和檢測的正對照。
b. 凝集素結合特異性應在 0.3 mol/L 抑制性糖（表 25-1 ) 濃度下進行親和檢測驗證。
( 5 ) 可用的抗 O-糖苷抗體有：抗
AGP 抗體：LM2 [ 31，32] 、JIM 4、JIM 13、JIM 15 [32]、JIM 8 [33] 、JIM 14、JIM 15
和 JIM 16 [34]；抗伸展蛋白抗體：LM 1 [ 35 ] 、JIM 11、 JIM 12、JIM 20 [36] 和 JIM19 [
37] 。
( 6 ) 免疫檢測的 N-糖苷特異性對照：應驗證血清對連接在被測蛋白的 N-糖苷的特異性，可在免疫檢測前先對印跡進行溫和的高碘酸鹽氧化處理，溫和高碘酸鹽處理會氧化糖苷，并消除糖蛋白上抗苷抗體的識別位點。剩下的信號都是蛋白骨架抗體識別的結果 [ 38 ]。
a. 經明膠飽和處理后，將蛋白質印跡膜浸泡在 100 mmol/L 含 100 mmol/L 過碘酸鈉的乙酸鈉緩沖液（pH 4.5 ) 中，室溫下黑暗處理 1 h，30 min 后更換一次浸泡液。
b. 將蛋白質印跡膜浸泡在含 50 mmol/L 硼氫化鈉的 PBS 緩沖液中，室溫下處理 30 min。
c. 用 TBS 漂洗蛋白質印跡膜，用含 1% 凝膠 的 TBS 浸泡蛋白質印跡膜 15 min，進行如 25. 3. 2 節 1. 1 ) 所述的免疫檢測實驗。
( 7 ) 巖藻糖或者木糖的特異性對照：有些蛋白質可被用作 N-糖苷免疫檢測的正對照，源自蜂毒的磷脂酶含有 α-1-3巖藻糖殘基，不含 β-1-2 木糖。源自玉米 的 PHA-L ( 植物血凝素 L ) 和重組抗生物素蛋白是既含 β-1-2 木糖，也含 α-1-3 巖藻糖的糖蛋白 [ 18，40 , 41 ] 。
( 8 ) 這里介紹的方法需要 1 mg 蛋白質，較少的蛋白質使用量也適用。
( 9 ) 用肌醇做內標。
( 10 ) 由于還原性氨化反應是一種激烈的處理，可能會發生一些蛋白質修飾。為此，有時候最好酶切去除 O-糖苷。
( 11 ) 總的來說，我們實驗室不使用化學處理解離糖蛋白上的N - 糖苷。
( 12 ) Nonidet P40 的作用是結合游離的 SDS。
( 13 ) 需要 60~80 mg 的可溶油菜籽蛋白作為初始材料，制備足夠跑一張 2D 凝膠的糖蛋白。
( 14 ) α-甲基甘露糖是刀豆蛋白 A 的配體，它將替換固定化凝集素上的糖蛋白。
( 15 ) 在洗脫親和色譜柱時，我們觀察到一個重要的解離物質刀豆蛋白 A。這一解離物質污染了糖蛋白制備物，迫使我們必須同時跑一張分析用 2D 凝膠，另一張 2D 凝膠只上樣刀豆蛋白 A。染色后，我們選擇只在分析用 2D 凝膠上出現的點，棄除同時在這兩張 2D 凝膠上出現的點。
( 16 ) 150 ml 生長 6 天的擬南芥 cgl 突變體細胞培養物大約相當于 10 g 植物材料。
( 17 ) 細胞壁結合蛋白的去除是純化的第一步，因為這些蛋白不含 O-位 N-乙酰葡糖胺。如果需要就應將這一純化步驟包括在這個方法中。
( 18 ) 自由 O-位 N-乙酰葡糖胺是 WGA 的配體，它將替換固定凝集素上的糖蛋白。
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