Kozma Eszter, Paci Giulia, Estrada Girona Gemma, Lemke Edward A, Kele Péter
Chemical Biology Research Group, Research Centre for Natural Sciences, Institute of Organic Chemistry, Hungarian Academy of Sciences, Magyar tudósok krt 2, Budapest, 1117, Hungary.
Structural and Computational Biology Unit & Cell Biology and Biophysics Unit, European Molecular Biology Laboratory, Meyerhofstrasse 1, Heidelberg, 69117, Germany.
Methods Mol Biol. 2018;1728:337-363. doi: 10.1007/978-1-4939-7574-7_22.
Tetrazine-bearing fluorescent labels enable site-specific tagging of proteins that are genetically manipulated with dienophile modified noncanonical amino acids. The inverse electron demand Diels-Alder reaction between the tetrazine and the dienophile fulfills the criteria of bioorthogonality allowing fluorescent labeling schemes of live cells. Here, we describe the detailed synthetic and labeling protocols of a near infrared emitting siliconrhodamine-tetrazine probe suitable for super-resolution imaging of residue-specifically engineered proteins in mammalian cells.
带有四嗪的荧光标记能够对通过亲双烯体修饰的非天然氨基酸进行基因操作的蛋白质进行位点特异性标记。四嗪与亲双烯体之间的逆电子需求狄尔斯-阿尔德反应满足生物正交性标准，从而实现活细胞的荧光标记方案。在此，我们描述了一种近红外发射硅罗丹明-四嗪探针的详细合成和标记方案，该探针适用于对哺乳动物细胞中残基特异性工程化蛋白质进行超分辨率成像。
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