Compared to other proteomics methods, 2-D gel electrophoresis (2-D PAGE) is unique for providing several kinds of valuable information about any given protein in a single reaction or in comparative analysis (Rabilloud 2000). Additionally, 2-D PAGE is most valuable in evaluation of mixed protein samples, compared to other protein identification methods, such as Western blotting, which specifically characterize a single protein in every single assay. The information obtained from 2-D PAGE consists of the experimental isoelectric point (PI) and molecular weight (MW) of proteins, prediction of post-translational modifications, up and down-regulation of proteins and emergence of newly synthesized proteins or possible loss and degradation. Furthermore, in-tandem usage of 2-D with up-stream methods like immunoprecipitation, and also down-stream methods like Western blotting linked to mass spectrometry, is able to yield even more data about any protein under investigation.
In recent years, 2-D reference maps have been considered to be of high significance as evidenced by their central position in proteomics databases (e.g. ExPASy)( http://www.expasy.ch/ ). Using these databases helps researchers to trace any changes occurring in the mobility of a protein following exposure of a given cell to a defined stimulus. The advances in 2-D methods along with the progress in image analysis software and need to compare the protein profile of different species, cell lines or even different organelles, all have contributed for some protein databases such as ExPASy to dedicate a major part to 2-D PAGE data.
During last three decades 2-D gel electrophoresis has been the core approach in classic proteomics studies and many improvements have been implemented (Issaq and Veenstra 2008). However, 2-D PAGE is not a straightforward method and there are still many discrepancies in the 2-D maps from sample to sample or between results published by different investigators. Because of subtle changes in experimental condition, protein pattern of two successive experiments on 2-D gel usually cannot be fully duplicated, especially when obtained from primary cells.
Introduction of immobilized pH gradient (IPG) gels by Bejellqvist et al.(Bjellqvist, Ek et al. 1982) has significantly improved the reproducibility and resolution of 2-D maps in recent years. However, standardizing the process to get reproducible 2-D PAGE maps in different labs is highly important for future investigations.
Reproducibility of 2-D PAGE is highly dependent on efficacy of sample preparation. Presence of contaminants, such as salt, nucleic acids and lipids, in the protein sample usually interfere with the generation of satisfactory results. Furthermore, the limitation of the quantity of protein that can be loaded on DryStrip, is considered as another major disadvantage of 2-D PAGE that makes protein characterization and identification difficult.
