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J Biomol Tech. 2007 February; 18(1): 62.
PMCID: PMC2291959

P179-M A Verification of Novel Experimental Design for Comparative Two-Dimensional Gel Analysis: Pre-Labeled Samples Fractionated on Anion Exchange Column Followed by 2D DIGE

Abstract

A common issue in Proteomics is that analytical methods of separation do not have sufficient resolving power to deal with the tremendous sample complexity normally associated with a proteomics experiment. While 2D electrophoresis is superior to other methods in this respect, proteins can still co-migrate. Different methods of pre-fractionation have been employed in the past to rectify this problem. Recently, we demonstrated that E. coli samples pre-labeled with Cy2, Cy3, and Cy5 and fractionated on an anion exchange column resulted in reproducible fractions, without introducing any bias associated with the pre-labeling. To further verify the reliability of this pre-fractionation method for quantitative purposes, we spiked four different amounts of 5 proteins (not native to E. coli) into the sample. The fractions were run on an anion exchange column and subsequently resolved with 2D electrophoresis. The DIGE gels were analyzed with DeCyder 2D v. 6.5. The results from this study show a very close correlation between the expected theoretical results and the actual experimental results.


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