Aim:To develop a simple method for monitoring protein localization of epidermalgrowth factor(EGF)in living cells.Methods:Enhanced green fluorescent protein(EGFP)was used as an autofluorescent tag to label EGF ligands.SDS-PAGE andWestern blot analysis were used to detect the expression of the EGFP-tagged EGF(EGFP-EGF)protein.The cell-binding and internalization activity of EGFP-EGFwere analyzed by fluorescence-activated cell sorting(FACS)and confocal micro-scopy.Results:EGFP-EGF protein was expressed in Escherichia coli and purified.A cell-binding assay demonstrated that the EGFP-EGF protein could bind effi-ciently to the cells expressing EGFR.The binding and internalization of EGFP-EGFcan be visualized even at a very low concentration under confocal microscopy.The FACS-based assay for internalization activity indicated the accumulation ofinternalized EGFP-EGF over time.Furthermore,the results of the competitionassay indicated its EGFR binding specificity.Using such a method,it does notneed to label EGF with chemicals and avoid light in the experimental process.Conclusion:The fusion protein EGFP-EGF has several characters including highsensitivity,stability and convenience for manipulation,and is a powerful tool forthe study of EGF endocytosis. Aim: To develop a simple method for monitoring protein localization of epidermalgrowth factor (EGF) in living cells. Methods: Enhanced green fluorescent protein (EGFP) was used as an autofluorescent tag to label EGF ligands. SDS-PAGE and Western blot analysis were used to detect the expression of the EGFP-tagged EGF (EGFP-EGF) protein.The cell-binding and internalization activity of EGFP-EGFwere analyzed by fluorescence-activated cell sorting (FACS) and confocal micro-scopy. Results: EGFP-EGF protein was expressed in Escherichia coli and purified. A cell-binding assay demonstrated that the EGFP-EGF protein could bind effi-ciently to the cells expressing EGFR. The binding and internalization of EGFP-EGFcan be visualized even at a very low concentration under confocal microscopy. The FACS-based assay for internalization activity indicated the accumulation of internalized EGFP-EGF over time. Future and the results of the competition assay that its EGFR binding specificity. Use such a method, it does not need to label EGF with chemicals and avoid light in the experimental process. Confluence: The fusion protein EGFP-EGF has several characters including high sensitivity, stability and convenience for manipulation, and is a powerful tool forthe study of EGF endocytosis.