Kawashima H, Murata T, Yamamoto K, Tateishi A, Irimura T, Osawa T
Division of Chemical Toxicology and Immunochemistry, Faculty of Pharmaceutical Sciences, University of Tokyo.
J Biochem. 1992 May;111(5):620-2. doi: 10.1093/oxfordjournals.jbchem.a123807.
A simple method for the release of oligosaccharides from glycoproteins separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) has been developed. Asialo-alpha 1-acid glycoprotein, which was tritiated at the nonreducing terminal D-galactopyranosyl residue by reduction with sodium borotritide after incubation with D-galactose oxidase, was used as a model compound. After electrophoretic separation of the glycoprotein, oligosaccharides were released by the use of a gas-phase hydrazinolysis apparatus. In the first method, the gel was stained with Coomassie Blue and the glycoprotein together with the gel was directly subjected to gas-phase hydrazinolysis after removal of water in a P2O5 desiccator. The recovery of released oligosaccharides was 25.9 +/- 2.4%, based on the amount of the glycoprotein loaded on the gel within the range of 3.5-28.5 micrograms. In the second method, the glycoprotein was electroblotted onto an Immobilon transfer membrane and was visualized by staining with Coomassie Blue. A small piece of the membrane with the corresponding band was cut out, dried in a desiccator and subjected to gas-phase hydrazinolysis. In this case, the recovery of released oligosaccharides was 15.2 +/- 1.0%. These procedures, particularly the first one, should be widely applicable for the isolation of oligosaccharides from glycoproteins separated by SDS-PAGE.
已开发出一种从经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（SDS-PAGE）分离的糖蛋白中释放寡糖的简单方法。去唾液酸-α1-酸性糖蛋白，在与D-半乳糖氧化酶孵育后，通过用硼氢化三钠还原在非还原末端D-吡喃半乳糖残基处进行了氚标记，用作模型化合物。糖蛋白经电泳分离后，使用气相肼解装置释放寡糖。在第一种方法中，凝胶用考马斯亮蓝染色，糖蛋白与凝胶一起在P2O5干燥器中除去水分后直接进行气相肼解。在所加凝胶的糖蛋白量为3.5 - 28.5微克范围内，释放的寡糖回收率为25.9±2.4%。在第二种方法中，糖蛋白电印迹到Immobilon转移膜上，并用考马斯亮蓝染色进行可视化。切下带有相应条带的一小片膜，在干燥器中干燥并进行气相肼解。在这种情况下，释放的寡糖回收率为15.2±1.0%。这些方法，特别是第一种方法，应广泛适用于从经SDS-PAGE分离的糖蛋白中分离寡糖。
