大学化学 >> 2022, Vol. 37 >> Issue (5): 2111072.doi: 10.3866/PKU.DXHX202111072

所属专题: 第2届全国大学生化学实验创新设计竞赛

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蛋白质中总巯基的测定及标记

张保新, 范卓, 于晨昕, 刘喆, 沈永雯, 惠新平, 房建国()   

  • 收稿日期:2021-11-24 录用日期:2021-12-07 发布日期:2022-04-06
  • 通讯作者: 房建国 E-mail:fangjg@lzu.edu.cn
  • 作者简介:房建国, Email: fangjg@lzu.edu.cn
  • 基金资助:
    基金委国家基础科学人才培养基金(J1030416);兰州大学教学研究项目(201118);基础学科拔尖学生培养计划2.0研究课题(20212090)

Labeling and Determination of Total Sulfhydryl Content in Protein

Baoxin Zhang, Zhuo Fan, Chenxin Yu, Zhe Liu, Yongwen Shen, Xinping Hui, Jianguo Fang()   

  • Received:2021-11-24 Accepted:2021-12-07 Published:2022-04-06
  • Contact: Jianguo Fang E-mail:fangjg@lzu.edu.cn

摘要:

生物硫醇的检测与蛋白质荧光标记是当今科学研究的热门领域。本实验使用传统的Ellman法及Naph-EA-Mal荧光探针标记法对牛血清白蛋白(Bovine Serum Albumin, BSA)的巯基进行检测,并利用该探针实现了细胞内巯基的原位标记,使学生对传统方法和前沿热点均有较好的掌握和了解。通过本实验的开展,学生不仅可以清楚了解蛋白三维结构对其所含基团的反应活性的影响,而且可以从化学层面理解硫醇类探针的工作原理及活细胞中生物硫醇的原位成像。实验内容涵盖蛋白质还原、变性、生物大分子与小分子的分离、生物大分子的化学修饰以及活细胞荧光成像等,综合性强且容易操作。教学内容可以实现模块化,便于不同学校根据条件选取相应的教学内容。教学实践不仅可以让学生复习巩固化学生物学知识,也帮助学生了解前沿交叉学科热点,同时在虚拟实验平台开设了补充实验,使得教学更加立体化。

关键词: 蛋白质巯基, Ellman法, 荧光探针, 活细胞成像, 蛋白质的化学修饰

Abstract:

Biological sulfhydryl detection and protein labeling are critical in current scientific research. In this study, the traditional Ellman method and the newly developed Naph-EA-Mal fluorescent probe were used to quantify sulfhydryl groups in bovine serum albumin, thus giving students a better understanding of both traditional methods and cutting-edge technology. The content of sulfhydryl can be detected in situ through measuring the fluorescence intensity. In addition, students gain knowledge regarding the mechanism of thiol probes and the influence of the protein's three-dimensional structure on the reactivity of the sulfhydryl groups within the protein. The experiment covers basic technology of treating cells and different methods of treating biomacromolecules. These methods are comprehensive, modularized, and easy to implement. This lab course will help students obtain cutting-edge chemical biology knowledge. In addition, the experiments on the virtual experiment platform could further help students learn and understand the principles and operational methods used in this experiment.

Key words: Protein sulfhydryl group, Ellman method, Fluorescent probe, Live cell imaging, Protein chemical modifications

MSC2000: 

  • G64