Photocrosslinking O-GlcNAcylated Proteins to Neighboring Biomolecules.
Photocrosslinking O-GlcNAcylated Proteins to Neighboring Biomolecules.
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DOI：
10.1002/cpz1.201
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发表时间：
2021-07
期刊：
影响因子：
--
通讯作者：
Kohler JJ
中科院分区：
文献类型：
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作者：
Capota E;Wu H;Kohler JJ
该方案能够鉴定O-GlcNAc酰化蛋白质的相互作用伴侣。该方法涉及将二氮丙环光交联剂引入活细胞内的O-GlcNAc修饰上。光交联剂被紫外光激活，在O-GlcNAcylated蛋白质和邻近分子之间产生共价交联。结合配偶体可以通过免疫印迹或蛋白质组学质谱法进一步表征。使用光交联剂的益处包括捕获低亲和力结合相互作用的能力和选择性靶向目标蛋白质的O-GlcNAc酰化形式的相互作用配偶体的能力。基本方案1：基础方案2：免疫印迹以评估O-GlcNDAZ交联支持方案1：从细胞裂解物检测UDP-GlcNDAZ
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. Basic Protocol 1: In-cell production and crosslinking of O-GlcNDAzylated proteins Basic Protocol 2: Immunoblot to assess O-GlcNDAz crosslinking Support Protocol 1: Detection of UDP-GlcNDAz from cell lysates