Department of Early Discovery Biochemistry, Genentech, South San Francisco, California, USA.
Nat Protoc. 2014 Nov;9(11):2607-23. doi: 10.1038/nprot.2014.179. Epub 2014 Oct 9.
Dissecting the subcellular distribution of a fatty-acylated protein is key to the understanding of the molecular mechanisms regulating protein movement and function in a cell. This protocol describes how to perform single-cell imaging of palmitoylation in a fatty-acylated protein of interest with high sensitivity using click chemistry, proximity ligation and fluorescence microscopy. The initial steps in this protocol involve optimization of conditions for (i) metabolic incorporation of an alkynyl analog of palmitic acid into cellular proteins coupled with click chemistry and (ii) detecting a specific protein of interest with primary antibodies using automated fluorescence microscopy, followed by (iii) imaging palmitoylation of the target fatty-acylated protein of interest, such as Wnt, Sonic Hedgehog or H-Ras. Furthermore, we outline strategies for imaging specific fatty-acylated proteins with subcellular organelles and/or total proteome palmitoylation, and we discuss special considerations that need to be given depending on the experimental design. The use of clickable fatty acids with proximity ligation may have promising applications to the investigation of fatty acylation cell biology. The entire protocol takes ∼3 weeks to complete.
解析脂肪酸酰化蛋白的亚细胞分布对于理解调节细胞内蛋白质运动和功能的分子机制至关重要。本方案描述了如何使用点击化学、邻近连接和荧光显微镜以高灵敏度对感兴趣的脂肪酸酰化蛋白中的棕榈酰化进行单细胞成像。该方案的初始步骤包括优化以下条件：（i）将丙炔基棕榈酸类似物代谢掺入细胞蛋白中，然后进行点击化学反应，（ii）使用自动化荧光显微镜用初级抗体检测特定的感兴趣蛋白，然后（iii）对感兴趣的目标脂肪酸酰化蛋白（如 Wnt、Sonic Hedgehog 或 H-Ras）进行棕榈酰化成像。此外，我们还概述了针对细胞器和/或整个蛋白质组棕榈酰化的特定脂肪酸酰化蛋白成像的策略，并根据实验设计讨论了需要考虑的特殊注意事项。使用具有邻近连接的可点击脂肪酸可能对脂肪酸酰化细胞生物学的研究具有广阔的应用前景。整个方案大约需要 3 周时间才能完成。
