Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro‐Q Emerald 488 dye, a fluorescent periodate Schiff‐base stain

Hart, Courtenay; Schulenberg, Birte; Steinberg, Thomas H.; Leung, Wai‐Yee; Patton, Wayne F.

doi:10.1002/elps.200390069

Cited by 101 publications

(77 citation statements)

References 21 publications

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“…Proteins were visualized with colloidal Coomassie Brilliant Blue R-250 staining reagent, carbohydrates were visualized with Pro-Q Emerald 300 fluorescent stain (Invitrogen) (21), and the gels were imaged at 700 nm using the Odyssey imaging system (LI-COR) and at 300 nm using the Infinity-3000 (Vilber-Lourmat) apparatus, respectively. Tank blotting of (glyco)proteins to a polyvinylidene difluoride membrane was performed as described previously (22).…”

Section: Methodsmentioning

confidence: 99%

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

Posch

Pabst

Brecker

et al. 2011

Journal of Biological Chemistry

188

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Section: Methodsmentioning

confidence: 99%

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

Posch

Pabst

Brecker

et al. 2011

Journal of Biological Chemistry

188

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“…The main limitation to the use of Pro-Q Emerald 300 is that it cannot be used with laserbased gel scanners. This disadvantage led to the development of a new fluorescent glycoprotein stain, Pro-Q Emerald 488 [213]. Detection sensitivity of glycosylated protein depends greatly on carbohydrate content (α 2 -macroglobulin (9-10% CHO), glucose oxidase (12-13% CHO) and fetuin (22% CHO)-9.4 ng; α 1 -acid glycoprotein (38-42% CHO)-4.7 ng; avidin (7% CHO) and OVA (3-4% CHO)-18.8 ng).…”

Section: Glycosylated Proteinsmentioning

confidence: 99%

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

2010

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Proteomics research relies heavily on visualization methods for detection of proteins separated by polyacrylamide gel electrophoresis. Commonly used staining approaches involve colorimetric dyes such as Coomassie Brilliant Blue, fluorescent dyes including Sypro Ruby, newly developed reactive fluorophores, as well as a plethora of others. The most desired characteristic in selecting one stain over another is sensitivity, but this is far from the only important parameter. This review evaluates protein detection methods in terms of their quantitative attributes, including limit of detection (i.e., sensitivity), linear dynamic range, inter-protein variability, capacity for spot detection after 2D gel electrophoresis, and compatibility with subsequent mass spectrometric analyses. Unfortunately, many of these quantitative criteria are not routinely or consistently addressed by most of the studies published to date. We would urge more rigorous routine characterization of stains and detection methodologies as a critical approach to systematically improving these critically important tools for quantitative proteomics. In addition, substantial improvements in detection technology, particularly over the last decade or so, emphasize the need to consider renewed characterization of existing stains; the quantitative stains we need, or at least the chemistries required for their future development, may well already exist.

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“…These stains offer broad dynamic ranges (500-1000-fold) for quantification, and do not interfere with MS analyses for protein identification [134][135][136][137].…”

Section: Post-translational Modificationmentioning

confidence: 99%

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

Penque

2009

Proteomics Clinical Apps

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The proteome project, initiated in 1995, was made possible by 2-DE combined with MS. The project main objective was and remains the identification of all proteins expressed by a cell, tissue or organism in a given time and condition. Following this objective, the global profiling of proteins in health versus pathological state by the 2-DE/MS-based proteomic approach has contributed to the elucidation of the basic mechanisms of disease by discovering candidate disease biomarkers and disease targets for new drug development. This review will briefly summarize the historical evolution of 2-DE up to today, and review 2-DE/MS technology and its specific methods of study of immunoresponse (immunoproteomics), PTM of proteins, complex proteinprotein interactions (interactome), the proteome of cell membrane and intracellular proteome turnover in disease biomarker discovery. 2-DE historical evolutionGlobal profiling of proteins in healthy versus pathological states is a strategy to discover biomarkers for early diagnostics, disease progression, treatment response and novel targets for therapeutic drugs development. The idea of making an inventory of all the proteins in a cell, tissue or organism with normal or abnormal physiology, was (re)initiated in the middle of the 1990s with the proteome project [1]. Since then, many technologies to make the analysis of the proteome a reality have been united.The perfect technological 'marriage' that made the proteome project feasible was between high resolution 2-DE for separation of large numbers of proteins and MS for identification and characterization of the proteins displayed on this gel [2]. However, before this happy union occured, science and technology devoted to protein study had a long journey until 2-DE and MS combination was possible (Fig. 1). It started in the last century, in 1937, when Tiselius [3] introduced electrophoresis technique as a modification of Reiner's early concept (1927) [4] to analyse a complex mixture of proteins such as serum proteins. Only 20 years later, in 1959, electrophoresis on a gel support formed by crosslinked polymerization of Cyanogum 41, a commercial name for two organic monomers, acrylamide and the crosslinking agent, N,N1-methylenebisacrylamide, was shown to be useful and was named PAGE by Raymond and Weintraub [5]. The adaptation of polyacrylamide gel to IEF in a stable pH gradient, developed by Svensson in 1962 [6], was possible by the end of the 1960s [7][8][9]. About this time, the 2-D by combining IEF to separate undenatured proteins according to their charge and gradient SDS-PAGE for a second separation, according to their molecular mass, was for the first time introduced by Kenrick and Margolis (1970) [10]. Only in 1975, was IEF in denaturing conditions, as known today, used to follow the 2-D PAGE and it was described in three independent works [11][12][13]. The most powerful demonstraCorrespondence: Dr. Deborah Penque, Laboratório de Proteó-mica, Departamento de Genética, Instituto Nacional de Saúde, Dr. Ricardo Jorge, I.P., A...

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Detection of glycoproteins in polyacrylamide gels and on electroblots using Pro‐Q Emerald 488 dye, a fluorescent periodate Schiff‐base stain

Cited by 101 publications

References 21 publications

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

Characterization and Scope of S-layer Protein O-Glycosylation in Tannerella forsythia

Quantitative proteomics: assessing the spectrum of in-gel protein detection methods

Two‐dimensional gel electrophoresis and mass spectrometry for biomarker discovery

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