Overviewforworkflow实验流程概述.PDFVIP

Ludger Document #Release-Labelling-2AB-Method-v1.0.doc Ludger Standard N-glycan Release, 2AB labelling, Clean-up and HILIC-(U)HPLC Methods Ludger 标准的N-多糖释放, 2AB标记，纯化和HILIC-(U)HPLC方法 This document provides the details of one standard set of methods used at Ludger for glycan release and profiling of 2AB labelled N-glycans. This is the typical set of methods that we would use to analyse N-glycans from mammalian derived glycoproteins. 本文提供了标准的Ludger多糖释放和2AB标记的N-多糖分析方法的细节 。这是我们分析哺乳动物衍生的糖蛋白中的N-多糖的典型方法。 This document should be used in conj unction with the individual product guides that can be located under the Product Categories at: /products/该文件应该与相应的产品使用指南联合使用,可以在产品类别中 下载:/products/ Overview for workflow 实验流程概述 Stage 1 第 1 阶段: Release N-glycans 释放N-多糖 Stage 2 (optional) 第2 阶段(可选的): Conversion of released Nglycans to aldoses 转换释放的N-多糖成 醛糖 Stage 3第3 阶段: Clean-up纯化 Stage 4第4 阶段: 2-AB labeling 2-AB标记 Stage 5第5 阶段: Post-labelling clean-up标记后纯化 Stage 6第6阶段: HILIC-HPLC or HILIC-UPLC analysis. HILIC-HPLC or HILIC-UPLC分析 Method Details 方法细节 Stage 1第 1 阶段: Release N-glycans释放N-多糖 Reagents: E-PNG-01; Pure water (resistivity above 18 MΩ-cm, particle free (0.22 µm), TOC 10 ppb) is used throughout. 试剂:E-PNG-01;纯水(电阻率高于18 MΩ-cm，不含粒子( 0.22µm), TOC 10 ppb) 通篇使用。 1. Add up to 200μg of glycoprotein to an Eppendorf tube. Adj ust to 35 μl final volume with de-ionized water. 加200μg糖蛋白到一个微量离心管。用去离子水调整到最后体积为35μl 。 Add 10 μl5x Reaction Buffer pH 7 and 2.5 μlof Denaturation Solution. Heat at 100°C for 5 minutes. 加10μlpH 7 的5 倍反应缓冲液和2.5μl变性液。加热5分钟至100°C 。 2. Cool. Add 2.5 μl of Triton X-100 and mix. 冷却：加2.5μl曲通X-100然后混合。 3. Add 2.0 μl of PNGase F to the reaction. Incubate 3 to 24 hours