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Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence imaging.

Authors :
Yu Z
Cao Y
Tian Y
Ji W
Chen KE
Wang Z
Ren J
Xiao H
Zhang L
Liu W
Fan L
Zhang Q
Cao C
Source :
Analytica chimica acta [Anal Chim Acta] 2024 Feb 22; Vol. 1291, pp. 342219. Date of Electronic Publication: 2024 Jan 05.
Publication Year :
2024

Abstract

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.<br />Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.<br /> (Copyright © 2024 Elsevier B.V. All rights reserved.)

Details

Language :
English
ISSN :
1873-4324
Volume :
1291
Database :
MEDLINE
Journal :
Analytica chimica acta
Publication Type :
Academic Journal
Accession number :
38280790
Full Text :
https://doi.org/10.1016/j.aca.2024.342219