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Updated: Jul 4, 2025
Fluorescent Silver Staining of Proteins in Polyacrylamide Gels
Published on: April 21, 2019
Real-time and quantitative protein detection via polyacrylamide gel electrophoresis and online intrinsic fluorescence
Zixian Yu1, Yiren Cao2, Youli Tian3
1School of Sensing Science and Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, 200240, China.
Insights
A new online intrinsic fluorescence imaging (IFI) method for polyacrylamide gel electrophoresis (PAGE) enables real-time, label-free protein detection. This PAGE-IFI technique enhances sensitivity and resolution for quantitative protein analysis in molecular biology labs.
Area of Science:
- Biochemistry and Molecular Biology
- Analytical Chemistry
- Biophysical Chemistry
Background:
- Intrinsic protein fluorescence offers label-free, stain-free analysis of proteins in their native state.
- Conventional polyacrylamide gel electrophoresis (PAGE) detection methods are often tedious and time-consuming.
- Existing intrinsic fluorescence detection methods are incompatible with online PAGE or standard slab gels.
Purpose of the Study:
- To develop a real-time, quantitative protein detection method using intrinsic fluorescence imaging (IFI) compatible with standard slab polyacrylamide gel electrophoresis (PAGE).
- To overcome limitations of previous intrinsic fluorescence detection methods by enabling online detection with standard gels.
Main Methods:
- Developed a PAGE-IFI method integrating deep-UV light source and a semi-open gel electrophoresis apparatus (GEA) for online imaging of standard slab gels.
- Investigated light source arrangement for large imaging areas and designed GEA for low background noise.
- Optimized PAGE run endpoint based on real-time protein migration monitoring.
Main Results:
- Achieved real-time monitoring of protein migration, improving intrinsic fluorescence imaging (IFI) sensitivity by determining optimal PAGE endpoint.
- Enhanced separation resolution by circumventing protein band broadening through online IFI.
- Demonstrated quantitative detection of bovine serum albumin (BSA) with a limit of detection (LOD) of 20 ng and a wide linear range (0.03–10 μg) due to high sample loading capacity.
Conclusions:
- The developed PAGE-IFI method provides a sensitive, quantitative, and label-free alternative for protein analysis.
- This method is compatible with standard slab gels and offers real-time monitoring, enhancing traditional PAGE techniques.
- PAGE-IFI holds promise for widespread adoption in molecular biology laboratories for efficient protein analysis.
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.
