蛋白质的非定点荧光标记——推荐一个化学生物学实验

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

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。

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.