大学化学 >> 2024, Vol. 39 >> Issue (4): 351-360.doi: 10.3866/PKU.DXHX202310010

化学实验 上一篇    下一篇

蛋白质的非定点荧光标记——推荐一个化学生物学实验

洪歆怡, 薛太玲, 徐洲, 谢恩蓉, 吴明凯, 王青青, 吴丽娜   

  1. 厦门大学化学化工学院, 化学国家级实验教学示范中心(厦门大学), 福建 厦门 361005
  • 收稿日期:2023-10-09 修回日期:2023-12-29 发布日期:2024-01-09
  • 通讯作者: 吴丽娜 E-mail:alina1222@xmu.edu.cn
  • 基金资助:
    2020年厦门大学教学改革研究项目

Non-Site-Specific Fluorescent Labeling of Proteins as a Chemical Biology Experiment

Xinyi Hong, Tailing Xue, Zhou Xu, Enrong Xie, Mingkai Wu, Qingqing Wang, Lina Wu   

  1. National Demonstration Center for Experimental Chemistry Education (Xiamen University), College of Chemistry and Chemical Engineering, Xiamen University, Xiamen 361005, Fujian Province, China
  • Received:2023-10-09 Revised:2023-12-29 Published:2024-01-09

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摘要： 本论文介绍了一项化学生物学实验，旨在利用异硫氰酸荧光素的异硫氰基与蛋白质氨基共价结合的原理，实现对体外蛋白(以牛血清白蛋白为例)和体内蛋白(以大肠杆菌总蛋白为例)的非定点荧光标记。实验采用凝胶过滤法作为后续纯化手段，并运用紫外-可见分光光度计、流式细胞术以及荧光显微镜对标记结果进行全面表征。该实验涵盖了多种基础和前沿实验技能，与科研工作中常见的抗体荧光标记技术密切相关，难度适中。通过参与这一实验，学生不仅能够深入理解化学生物学基础知识，提高综合实践能力，还能够拓宽科研视野并培养科研思维。

关键词: 非定点荧光标记, 蛋白质, 异硫氰酸荧光素, 化学生物学实验

Abstract: This paper presents a chemical biology experiment designed to exploit the principle of covalent binding between fluorescein isothiocyanate and protein amines. This process is aimed at achieving non-site-specific fluorescent labeling of both extracellular proteins, exemplified by bovine serum albumin, and intracellular proteins, using Escherichia coli total protein as a case study. Gel filtration chromatography is employed as a subsequent purification method, with comprehensive characterization of the labeling results performed using UV-Visible spectrophotometry, flow cytometry, and fluorescence microscopy. This experiment encompasses a range of foundational and cutting-edge laboratory skills. It is closely aligned with the commonly employed antibody fluorescence labeling techniques in scientific research, presenting a moderate level of complexity. Participation in this experiment allows students to gain a profound understanding of fundamental concepts in chemical biology, enhance their comprehensive practical skills, broaden their research perspectives and nurture scientific thinking.

Key words: Non-site-specific fluorescent labeling, Protein, Fluorescein isothiocyanate, Chemical biology experiment