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Use of a Fluorescent Internal Protein Standard to Achieve Quantitative Two-Dimensional Gel Electrophoresis

Use of a Fluorescent Internal Protein Standard to Achieve Quantitative Two-Dimensional Gel Electrophoresis

Asa M Wheelock, Dexter Morin, Matthew Bartosiewicz, Alan R Buckpitt

Proteomics. 2006 Mar;6(5):1385-98.

PMID: 16429456

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 (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.

Chemicals Related in the Paper:

Catalog Number	Product Name	Structure	CAS Number	Price
AP5873165	Sulforhodamine G		5873-16-5	Price

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