Lefebvre Tony, Drougat Ludivine, Olivier-Van Stichelen Stephanie, Michalski Jean-Claude, Vercoutter-Edouart Anne-Sophie
Unit of Structural and Functional Glycobiology, University of Lille 1, Villeneuve d'Ascq, France.
Methods Mol Biol. 2013;1022:147-59. doi: 10.1007/978-1-62703-465-4_12.
Since the discovery of O-GlcNAc modification (O-GlcNAcylation) 20 years ago, much attention has been given to OGT (O-GlcNAc transferase), the unique enzyme responsible for the nuclear and cytosolic O-GlcNAcylation processes. This review focuses on protocols that are routinely used to analyze OGT expression and activity. First are detailed techniques using rabbit polyclonal anti-OGT antibodies, namely, Western blot, (co-)immunoprecipitation, and immunofluorescence. We also describe the measurement of OGT activity by using synthetic peptides as acceptors and radiolabeled UDP-GlcNAc. Finally, a sensitive HPAEC-based technique to measure the cellular content of UDP-GlcNAc, the donor substrate of OGT, is described in detail.
自20年前发现O-连接的N-乙酰葡糖胺修饰（O-GlcNAcylation）以来，负责细胞核和细胞质O-GlcNAcylation过程的独特酶——O-GlcNAc转移酶（OGT）受到了广泛关注。本综述重点介绍了常用于分析OGT表达和活性的实验方案。首先详细介绍使用兔多克隆抗OGT抗体的技术，即蛋白质免疫印迹法、（共）免疫沉淀法和免疫荧光法。我们还描述了以合成肽为受体和放射性标记的UDP-GlcNAc来测量OGT活性的方法。最后，详细介绍了一种基于高效阴离子交换色谱-脉冲安培检测法（HPAEC）的灵敏技术，用于测量OGT的供体底物UDP-GlcNAc的细胞含量。
