Two‐dimensional gel electrophoresis

Abstract

Abstract Two‐dimensional gel electrophoresis (2DE) with immobilized pH gradients (IPGs) combined with protein identification by mass spectrometry (MS) is currently the workhorse for proteomics. In spite of new promising technologies that have emerged (MudPIT, stable isotope labeling, arrays), 2DE is currently the only technique that can be routinely applied for parallel quantitative expression profiling of large sets of complex protein mixtures such as whole cell lysates. 2DE couples isoelectric focusing (IEF) and sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) to resolve proteins according to two independent parameters, that is, isoelectric point (pI) in the first dimension and molecular mass (M r ) in the second. 2DE can resolve more than 5000 proteins simultaneously (∼2000 proteins routinely), and can detect <1 ng of protein per spot. Furthermore, it delivers a map of intact proteins, which reflects changes in protein expression level, isoforms or posttranslational modifications. This is in contrast to LC‐MS/MS‐based methods, which perform analysis on peptides, where M r and pI information is lost. Whatever technology is used, proteome analysis is technically challenging, because the number of different proteins expressed at a given time under defined biological conditions is likely to be in the range of at least several thousand in total cell lysates. In this article, we describe the 2DE‐MS workflow including the following topics: Sample preparation/solubilization, protein separation by 2DE with IPGs, protein detection and quantitation, computer‐assisted analysis of 2DE patterns, protein identification and characterization, and 2D protein databases.