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Use of a fluorescent internal protein standard to achieve quantitative two‐dimensional gel electrophoresis
Author(s) -
Wheelock Åsa M.,
Morin Dexter,
Bartosiewicz Matthew,
Buckpitt Alan R.
Publication year - 2006
Publication title -
proteomics
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.26
H-Index - 167
eISSN - 1615-9861
pISSN - 1615-9853
DOI - 10.1002/pmic.200402083
Subject(s) - quantitative proteomics , chemistry , fluorescence , alexa fluor , chromatography , quantitative analysis (chemistry) , normalization (sociology) , detection limit , proteomics , biochemistry , physics , quantum mechanics , sociology , anthropology , gene
2‐DE is a powerful separation method for complex protein mixtures. However, large intergel variations in spot intensity limit its use for quantitative proteomics studies. To address this issue, we developed a fluorescent internal protein standard for use in 2‐DE analysis. Protein samples are spiked with an Alexa‐labeled internal standard (ALIS) prior to separation with 2‐DE. Due to the high extinction coefficient of the Alexa‐fluor, incorporation of 0.1% of total protein is sufficient to allow visualization of the internal standard yet low enough to avoid interference in subsequent quantification and identification steps. Following 2‐DE, total proteins are visualized with fluorescent postelectrophoretic stains spectrally separated from ALIS. Four protein stains, Deep Purple, Sulforhodamine G, ruthenium II‐tris(bathophenanthroline disulfonate) (RuTBS), and SYPRO Ruby, including improved purification and staining protocols for RuTBS and ten‐fold dilutions of SYPRO Ruby were evaluated. All staining protocols were compatible with the ALIS method and had similar LODs (1–4 ng) and dynamic ranges (10 3 ). ALIS is a powerful normalization method for quantitative 2‐DE which avoids potential problems associated with dual spot migration patterns observed in the DIGE method. Furthermore, ALIS provides significantly improved normality in the distribution of spot abundance‐variance compared to normalization through division by the total spot volume.