Use of a fluorescent internal protein standard to achieve quantitative two-dimensional gel electrophoresis.
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.
Wheelock
Asa M
AM
Department of Molecular Biosciences, School of Veterinary Medicine, University of California, Davis, CA, USA. asa@para-docs.org
Morin
Dexter
D
Bartosiewicz
Matthew
M
Buckpitt
Alan R
AR
D000818:Animals; D002863:Chromogenic Compounds / Q000737:chemistry; D015180:Electrophoresis, Gel, Two-Dimensional / Q000295:instrumentation / Q000379:methods* / Q000592:standards*; D005456:Fluorescent Dyes / Q000138:chemical synthesis / Q000737:chemistry*; D008163:Luminescent Measurements; D008297:Male; D010618:Phenanthrolines / Q000737:chemistry; D011506:Proteins / Q000737:chemistry*; D040901:Proteomics / Q000379:methods; D011897:Random Allocation; D051381:Rats; D017207:Rats, Sprague-Dawley; D012015:Reference Standards; D017975:Ruthenium Compounds / Q000737:chemistry
D002863:Chromogenic Compounds; D005456:Fluorescent Dyes; D010618:Phenanthrolines; D011506:Proteins; D017975:Ruthenium Compounds; C017049:bathophenanthroline disulfonic acid