Freeze H H
The Burnham Institute, La Jolla, California, USA.
Curr Protoc Mol Biol. 2001 May;Chapter 17:Unit17.13A. doi: 10.1002/0471142727.mb1713as48.
Carbohydrate chain modifications are often used to monitor glycoprotein movement through the secretory pathway. This is because stepwise sugar-chain processing is unidirectional and generally corresponds to the forward or anterograde movement of proteins. This unit offers a group of techniques that will help analyze the general structure of carbohydrate chains on a protein and, therefore, oligosaccharide processing mileposts. The sugar chains themselves are not analyzed, but their presence and structure are inferred from gel mobility differences after one or more enzymatic digestions. This approach is most often used in combination with [35S]Met pulse-chase metabolic labeling protocols, but they can be applied to any suitably labeled protein (e.g., biotinylated or 125I-labeled).
碳水化合物链修饰常被用于监测糖蛋白在分泌途径中的移动。这是因为逐步的糖链加工是单向的，并且通常与蛋白质的正向或顺行移动相对应。本单元提供了一组技术，有助于分析蛋白质上碳水化合物链的总体结构，从而推断寡糖加工的里程碑。糖链本身并不进行分析，而是通过一次或多次酶消化后凝胶迁移率的差异来推断它们的存在和结构。这种方法最常与[35S]甲硫氨酸脉冲追踪代谢标记方案结合使用，但也可应用于任何经过适当标记的蛋白质（如生物素化或125I标记的蛋白质）。
