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