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Updated: Jul 4, 2026
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Chemiluminescence-based Assays for Detection of Nitric Oxide and its Derivatives from Autoxidation and Nitrosated Compounds
Published on: February 16, 2022
In-gel detection of S-nitrosated proteins using fluorescence methods
Nicholas J Kettenhofen1, Xunde Wang, Mark T Gladwin
1Department of Biophysics and Free Radical Research Center, Medical College of Wisconsin, Milwaukee, WI, USA.
Methods in Enzymology
|June 17, 2008
Summary
This study presents an improved biotin-switch method for detecting S-nitrosothiols. The modified technique uses copper ions and fluorescent dyes for direct, sensitive protein analysis.
Area of Science:
- Biochemistry
- Proteomics
- Molecular Biology
Background:
- Protein S-nitrosothiols are crucial in cellular signaling.
- The biotin-switch method is standard for detecting S-nitrosothiols but has limitations.
Purpose of the Study:
- To describe a modified biotin-switch assay for enhanced S-nitrosothiol detection.
- To enable direct in-gel visualization and comparative analysis of S-nitrosated proteins.
Main Methods:
- Selective reduction of S-nitrosothiols using copper ions and ascorbate.
- Direct labeling of reduced thiols with fluorescent cyanine dyes.
- In-gel detection and two-dimensional difference gel electrophoresis (2D-DIGE).
Main Results:
- The modified method allows direct visualization of S-nitrosated proteins in gels.
- Fluorescent labeling provides high sensitivity for detection.
- 2D-DIGE facilitates comparative proteomic studies of S-nitrosylation.
Conclusions:
- This modified assay offers a sensitive and direct approach for studying protein S-nitrosylation.
- The method simplifies the detection and analysis of S-nitrosated proteins.
- It is suitable for comparative proteomic studies and biomarker discovery.
