Martin Brent R
Department of Chemistry, University of Michigan, Ann Arbor, Michigan.
Curr Protoc Protein Sci. 2013 Sep 24;73:14.15.1-14.15.9. doi: 10.1002/0471140864.ps1415s73.
Methods to study protein S-palmitoylation dynamics have previously relied on metabolic labeling with [(14)C]palmitate, which requires additional safety precautions and long exposures. Nonradioactive alkynyl palmitate analogs have been developed for in-gel fluorescence detection and affinity purification. Cells metabolically labeled with the commercially available analog 17-octadynoic acid are lysed and then combined with azide-linked reporter tags for efficient conjugation by copper-catalyzed click chemistry in phosphate buffer. This approach has been demonstrated to label hundreds of endogenous palmitoylated proteins and is compatible with traditional pulse-chase methods. This protocol describes the reagents and procedures for labeling and detection of dynamic palmitoylation in mammalian cells.
此前，研究蛋白质S-棕榈酰化动力学的方法依赖于用[（14）C]棕榈酸进行代谢标记，这需要额外的安全预防措施和长时间曝光。已开发出用于凝胶内荧光检测和亲和纯化的非放射性炔基棕榈酸类似物。用市售类似物17-辛炔酸进行代谢标记的细胞被裂解，然后与叠氮化物连接的报告标签结合，以便在磷酸盐缓冲液中通过铜催化的点击化学进行高效共轭。该方法已被证明可标记数百种内源性棕榈酰化蛋白，并且与传统的脉冲追踪方法兼容。本方案描述了在哺乳动物细胞中标记和检测动态棕榈酰化的试剂和程序。
