Yu Zixian, Cao Yiren, Tian Youli, Ji Weicheng, Chen Ke-Er, Wang Zihao, Ren Jicun, Xiao Hua, Zhang Lu, Liu Weiwen, Fan Liuyin, Zhang Qiang, Cao Chengxi
School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China.
School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China; School of Chemistry and Chemical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China.
Anal Chim Acta. 2024 Feb 22;1291:342219. doi: 10.1016/j.aca.2024.342219. Epub 2024 Jan 5.
The detection of intrinsic protein fluorescence is a powerful tool for studying proteins in their native state. Thanks to its label-free and stain-free feature, intrinsic fluorescence detection has been introduced to polyacrylamide gel electrophoresis (PAGE), a fundamental and ubiquitous protein analysis technique, to avoid the tedious detection process. However, the reported methods of intrinsic fluorescence detection were incompatible with online PAGE detection or standard slab gel. Here, we fulfilled online intrinsic fluorescence imaging (IFI) of the standard slab gel to develop a PAGE-IFI method for real-time and quantitative protein detection. To do so, we comprehensively investigated the arrangement of the deep-UV light source to obtain a large imaging area compatible with the standard slab gel, and then designed a semi-open gel electrophoresis apparatus (GEA) to scaffold the gel for the online UV irradiation and IFI with low background noise. Thus, we achieved real-time monitoring of the protein migration, which enabled us to determine the optimal endpoint of PAGE run to improve the sensitivity of IFI. Moreover, online IFI circumvented the broadening of protein bands to enhance the separation resolution. Because of the low background noise and the optimized endpoint, we showcased the quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng. The standard slab gel provided a high sample loading volume that allowed us to attain a wide linear range of 0.03-10 μg. These results indicate that the PAGE-IFI method can be a promising alternative to conventional PAGE and can be widely used in molecular biology labs.
检测蛋白质固有荧光是研究天然状态下蛋白质的有力工具。由于其无需标记和染色的特性，固有荧光检测已被引入聚丙烯酰胺凝胶电泳（PAGE）这一基础且广泛应用的蛋白质分析技术中，以避免繁琐的检测过程。然而，已报道的固有荧光检测方法与在线PAGE检测或标准平板凝胶不兼容。在此，我们实现了标准平板凝胶的在线固有荧光成像（IFI），以开发一种用于实时定量蛋白质检测的PAGE - IFI方法。为此，我们全面研究了深紫外光源的布局，以获得与标准平板凝胶兼容的大成像区域，然后设计了一种半开放式凝胶电泳装置（GEA），用于支撑凝胶进行在线紫外照射和低背景噪声的IFI。因此，我们实现了对蛋白质迁移的实时监测，这使我们能够确定PAGE运行的最佳终点，以提高IFI的灵敏度。此外，在线IFI避免了蛋白条带变宽，从而提高了分离分辨率。由于背景噪声低且终点经过优化，我们展示了对牛血清白蛋白（BSA）的定量检测，检测限（LOD）为20 ng。标准平板凝胶提供了高样品上样量，使我们能够获得0.03 - 10 μg的宽线性范围。这些结果表明，PAGE - IFI方法可能是传统PAGE的一种有前景的替代方法，可广泛应用于分子生物学实验室。
