Optimization strategies for a fluorescent dye with bimodal excitation spectra: application to semiautomated proteomics

Wayne F. Patton; Kiera N. Berggren; Mary F. Lopez

doi:10.1117/12.424587

16 April 2001 Optimization strategies for a fluorescent dye with bimodal excitation spectra: application to semiautomated proteomics

Wayne F. Patton, Kiera N. Berggren, Mary F. Lopez

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Proceedings Volume 4264, Genomics and Proteomics Technologies; (2001) https://doi.org/10.1117/12.424587
Event: BiOS 2001 The International Symposium on Biomedical Optics, 2001, San Jose, CA, United States

Abstract

Facilities engaged in proteome analysis differ significantly in the degree that they implement automated systems for high-throughput protein characterization. Though automated workstation environments are becoming more routine in the biotechnology and pharmaceutical sectors of industry, university-based laboratories often perform these tasks manually, submitting protein spots excised from polyacrylamide gels to institutional core facilities for identification. For broad compatibility with imaging platforms, an optimized fluorescent dye developed for proteomics applications should be designed taking into account that laser scanners use visible light excitation and that charge-coupled device camera systems and gas discharge transilluminators rely upon UV excitation. The luminescent ruthenium metal complex, SYPRO Ruby protein gel stain, is compatible with a variety of excitation sources since it displays intense UV (280 nm) and visible (470 nm) absorption maxima. Localization is achieved by noncovalent, electrostatic and hydrophobic binding of dye to proteins, with signal being detected at 610 nm. Since proteins are not covalently modified by the dye, compatibility with downstream microchemical characterization techniques such as matrix-assisted laser desorption/ionization-mass spectrometry is assured. Protocols have been devised for optimizing fluorophore intensity. SYPRO Ruby dye outperforms alternatives such as silver staining in terms of quantitative capabilities, compatibility with mass spectrometry and ease of integration into automated work environments.

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Wayne F. Patton, Kiera N. Berggren, and Mary F. Lopez "Optimization strategies for a fluorescent dye with bimodal excitation spectra: application to semiautomated proteomics", Proc. SPIE 4264, Genomics and Proteomics Technologies, (16 April 2001); https://doi.org/10.1117/12.424587

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KEYWORDS

Proteins

Ruby

Visualization

Silver

Ultraviolet radiation

Bioalcohols

Image analysis

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