Two-Dimensional Electrophoresis: An Overview

Two-dimensional gel electrophoresis (2DE) separates proteins by molecular charge and molecular size. Proteins are first solubilised in a denaturing buffer containing a neutral chaotrope, a zwitterionic or neutral detergent, and a reducing agent. First-dimension isoelectric keywords, focusing, then subjects proteins to a high voltage within a pH gradient. The amphoteric nature of proteins means each migrates to the pH where the net molecular charge is zero. After equilibration, to ensure complete protein unfolding, the second dimension separates by molecular size. Each protein is therefore resolved at a unique isoelectric point/molecular size coordinate. After visualisation by staining proteome changes are revealed by gel image analysis, and protein spots of interest excised and identified by mass spectrometry sequence analysis combined with database comparison. Variations to this procedure include staining or radio-labelling prior to electrophoresis. Although 2DE does have limitations, the most significant being the resolution of membrane and/or hydrophobic proteins, the potential solutions offered by pre-fractionation or adjustments to the electrophoresis regimen mean this technique is likely to remain central to proteomic research.