Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis

Åsa M. Wheelock, Dexter Morin, Matthew Bartosiewicz, Alan R Buckpitt

Research output: Contribution to journalArticle

23 Scopus citations

Abstract

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 (103). 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.

Original languageEnglish (US)
Pages (from-to)1385-1398
Number of pages14
JournalProteomics
Volume6
Issue number5
DOIs
StatePublished - Mar 2006

Keywords

  • Alexa-labeled internal standard
  • Normalization
  • Quantitative two-dimensional gel electrophoresis

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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