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Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics

In this study, a commercially available fluorescent dye, Lissamine rhodamine B sulfonyl hydrazine (LRSH), was designed to specifically stain the glycoproteins in polyacrylamide gels. Through the periodate/Schiff base mechanism, the fluorescent dye readily attaches to glycoproteins and the fluorescen...

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Autores principales: Chiang, Yu-Hsuan, Wu, Yu-Jen, Lu, Ya-Ting, Chen, Kuan-Hung, Lin, Tzu-Chun, Chen, Yu-Kuang H., Li, Ding-Tzai, Shi, Fong-Ku, Chen, Ching-Chuan, Hsu, Jue-Liang
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Hindawi Publishing Corporation 2011
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184442/
https://www.ncbi.nlm.nih.gov/pubmed/21976968
http://dx.doi.org/10.1155/2011/780108
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author Chiang, Yu-Hsuan
Wu, Yu-Jen
Lu, Ya-Ting
Chen, Kuan-Hung
Lin, Tzu-Chun
Chen, Yu-Kuang H.
Li, Ding-Tzai
Shi, Fong-Ku
Chen, Ching-Chuan
Hsu, Jue-Liang
author_facet Chiang, Yu-Hsuan
Wu, Yu-Jen
Lu, Ya-Ting
Chen, Kuan-Hung
Lin, Tzu-Chun
Chen, Yu-Kuang H.
Li, Ding-Tzai
Shi, Fong-Ku
Chen, Ching-Chuan
Hsu, Jue-Liang
author_sort Chiang, Yu-Hsuan
collection PubMed
description In this study, a commercially available fluorescent dye, Lissamine rhodamine B sulfonyl hydrazine (LRSH), was designed to specifically stain the glycoproteins in polyacrylamide gels. Through the periodate/Schiff base mechanism, the fluorescent dye readily attaches to glycoproteins and the fluorescence can be simultaneously observed under either 305 nm or 532 nm excitation therefore, the dye-stained glycoproteins can be detected under a regular UV transilluminator or a more elegant laser-based gel scanner. The specificity and detection limit were examined using a standard protein mixture in polyacrylamide gels in this study. The application of this glycoprotein stain dye was further demonstrated using pregnancy urine samples. The fluorescent spots were further digested in gel and their identities confirmed through LC-MS/MS analysis and database searching. In addition, the N-glycosylation sites of LRSH-labeled uromodulin were readily mapped via in-gel PNGaseF deglycosylation and LC-MS/MS analysis, which indicated that this fluorescent dye labeling does not interfere with enzymatic deglycosylation. Hence, the application of this simple and specific dual-wavelength excitable dye staining in current glycoproteome research is promising.
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spelling pubmed-31844422011-10-04 Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics Chiang, Yu-Hsuan Wu, Yu-Jen Lu, Ya-Ting Chen, Kuan-Hung Lin, Tzu-Chun Chen, Yu-Kuang H. Li, Ding-Tzai Shi, Fong-Ku Chen, Ching-Chuan Hsu, Jue-Liang J Biomed Biotechnol Methodology Report In this study, a commercially available fluorescent dye, Lissamine rhodamine B sulfonyl hydrazine (LRSH), was designed to specifically stain the glycoproteins in polyacrylamide gels. Through the periodate/Schiff base mechanism, the fluorescent dye readily attaches to glycoproteins and the fluorescence can be simultaneously observed under either 305 nm or 532 nm excitation therefore, the dye-stained glycoproteins can be detected under a regular UV transilluminator or a more elegant laser-based gel scanner. The specificity and detection limit were examined using a standard protein mixture in polyacrylamide gels in this study. The application of this glycoprotein stain dye was further demonstrated using pregnancy urine samples. The fluorescent spots were further digested in gel and their identities confirmed through LC-MS/MS analysis and database searching. In addition, the N-glycosylation sites of LRSH-labeled uromodulin were readily mapped via in-gel PNGaseF deglycosylation and LC-MS/MS analysis, which indicated that this fluorescent dye labeling does not interfere with enzymatic deglycosylation. Hence, the application of this simple and specific dual-wavelength excitable dye staining in current glycoproteome research is promising. Hindawi Publishing Corporation 2011 2011-09-29 /pmc/articles/PMC3184442/ /pubmed/21976968 http://dx.doi.org/10.1155/2011/780108 Text en Copyright © 2011 Yu-Hsuan Chiang et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Methodology Report
Chiang, Yu-Hsuan
Wu, Yu-Jen
Lu, Ya-Ting
Chen, Kuan-Hung
Lin, Tzu-Chun
Chen, Yu-Kuang H.
Li, Ding-Tzai
Shi, Fong-Ku
Chen, Ching-Chuan
Hsu, Jue-Liang
Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics
title Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics
title_full Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics
title_fullStr Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics
title_full_unstemmed Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics
title_short Simple and Specific Dual-Wavelength Excitable Dye Staining for Glycoprotein Detection in Polyacrylamide Gels and Its Application in Glycoproteomics
title_sort simple and specific dual-wavelength excitable dye staining for glycoprotein detection in polyacrylamide gels and its application in glycoproteomics
topic Methodology Report
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3184442/
https://www.ncbi.nlm.nih.gov/pubmed/21976968
http://dx.doi.org/10.1155/2011/780108
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