Marter Kathrin, Kobler Oliver, Erdmann Ines, Soleimanpour Elaheh, Landgraf Peter, Müller Anke, Abele Julia, Thomas Ulrich, Dieterich Daniela C
Neuronal Plasticity and Communication, Institute for Pharmacology and Toxicology, Otto-von-Guericke-University Magdeburg, Magdeburg, Germany.
Research Group Neuralomics, Leibniz Institute for Neurobiology, Magdeburg, Germany.
Bio Protoc. 2019 Jan 20;9(2):e3142. doi: 10.21769/BioProtoc.3142.
Copper-catalyzed azide-alkyne-cycloaddition (CuAAC), also known as 'click chemistry' serves as a technique for bio-orthogonal, that is, bio-compatible labeling of macromolecules including proteins or lipids. Click chemistry has been widely used to covalently, selectively, and efficiently attach probes such as fluorophores or biotin to small bio-orthogonal chemical reporter groups introduced into macromolecules. In bio-orthogonal non-canonical amino acid tagging (BONCAT) and fluorescent non-canonical amino acid tagging (FUNCAT) proteins are metabolically labeled with a non-canonical, azide-bearing amino acid and subsequently CuAAC-clicked either to an alkyne-bearing biotin (BONCAT) for protein purification, Western blot, or mass spectrometry analyses or to an alkyne-bearing fluorophore (FUNCAT) for immunohistochemistry. In combination with mass spectrometry, these kinds of labeling and tagging strategies are a suitable option to identify and characterize specific proteomes in living organisms without the need of prior cell sorting. Here, we provide detailed protocols for FUNCAT and BONCAT click chemistry and the detection of tagged de novo synthesized proteins in .
铜催化的叠氮化物-炔烃环加成反应（CuAAC），也被称为“点击化学”，是一种用于生物正交反应的技术，即对包括蛋白质或脂质在内的大分子进行生物相容性标记。点击化学已被广泛用于将诸如荧光团或生物素等探针共价、选择性且高效地连接到引入大分子中的小生物正交化学报告基团上。在生物正交非天然氨基酸标记（BONCAT）和荧光非天然氨基酸标记（FUNCAT）中，蛋白质通过代谢被一种含叠氮基的非天然氨基酸标记，随后通过CuAAC反应与含炔基的生物素（用于蛋白质纯化、蛋白质免疫印迹或质谱分析）或与含炔基的荧光团（用于免疫组织化学）进行点击反应。与质谱联用，这些标记和标签策略是无需预先进行细胞分选即可识别和表征活生物体中特定蛋白质组的合适选择。在此，我们提供了FUNCAT和BONCAT点击化学以及检测新合成的标记蛋白质的详细方案。
