Department of Biochemistry, UT Southwestern Medical Center, Dallas, Texas.
Curr Protoc. 2021 Jul;1(7):e201. doi: 10.1002/cpz1.201.
This protocol enables identification of the interaction partners of O-GlcNAcylated proteins. The method involves the introduction of the diazirine photocrosslinker onto the O-GlcNAc modification within living cells. The photocrosslinker is activated by UV light to yield covalent crosslinking between O-GlcNAcylated proteins and neighboring molecules. The binding partners can be further characterized by immunoblot or proteomics mass spectrometry methods. The benefits of using the photocrosslinker include the capacity to trap low-affinity binding interactions and the ability to selectively target the interaction partners of the O-GlcNAcylated form of the protein of interest. © 2021 Wiley Periodicals LLC. Basic Protocol 1: In-cell production and crosslinking of O-GlcNDAzylated proteins Basic Protocol 2: Immunoblot analysis to assess O-GlcNDAz crosslinking Support Protocol: Detection of UDP-GlcNDAz from cell lysates.
本方案可用于鉴定 O-GlcNAc 修饰蛋白的相互作用伙伴。该方法涉及在活细胞内将叠氮化物光交联剂引入 O-GlcNAc 修饰中。光交联剂通过紫外光激活，在 O-GlcNAc 修饰蛋白与邻近分子之间产生共价交联。通过免疫印迹或蛋白质组学质谱方法可以进一步鉴定结合伙伴。使用光交联剂的好处包括能够捕获低亲和力的结合相互作用，以及能够选择性地针对感兴趣的蛋白的 O-GlcNAc 修饰形式的相互作用伙伴。© 2021 Wiley Periodicals LLC. 基本方案 1：细胞内 O-GlcNDAz 蛋白的产生和交联 基本方案 2：评估 O-GlcNDAz 交联的免疫印迹分析 支持方案：从细胞裂解物中检测 UDP-GlcNDAz。
