Abstract
Two-dimensional electrophoresis is an established method used to study differences in protein expression caused by, for example, a disease state or drug treatment. Conventional methods require the separation of one sample on each individual gel. This approach exposes the data to a high level of system variation, such as gel-to-gel variation caused by experimental factors. Quantification of protein differences can thus be uncertain and lead to false biological conclusions. Two alternatives to reduce this variation are (1) increase the number of replicates or (2) use the Ettan difference gel electrophoresis (DIGE) system. The Ettan DIGE system is a well-established technology in proteomics, which uses an internal standard for between-gel normalization. By pre-labeling samples prior to 2D electrophoresis with three spectrally resolvable CyDye DIGE Fluor dyes, electrophoretic co-migration of three protein samples on the same 2D gel is possible. This approach significantly reduces the number of replicate gels needed to ensure reproducibility and reliability of the differential expression analysis. Here, we present results from a differential expression analysis experiment performed with Escherichia coli samples grown under different conditions. We demonstrate that by using DIGE and the DeCyder 2D co-detection algorithm, the numbers of replicates are significantly reduced and the system variability is minimized compared to conventional electrophoresis with post-stained gels.