| Autor: |
Wheelock AM; Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA, USA. asa@para-docs.org, Morin D, Bartosiewicz M, Buckpitt AR |
| Jazyk: |
angličtina |
| Zdroj: |
Proteomics [Proteomics] 2006 Mar; Vol. 6 (5), pp. 1385-98. |
| DOI: |
10.1002/pmic.200402083 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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