Reduction and Alkylation of Proteins in 2D Gel Electrophoresis: Before or after Isoelectric Focusing?

Two-dimensional gel electrophoresis (2DE) is a well-developed, high-resolution technology to separate complex mixtures of proteins. This technique has been widely used for the past four decades, and has played a vital part in the developmental history of proteomics. The advantages and limitations of 2DE in proteomics research have been reviewed (e.g., Thelen and Peck, 2007; Oliveira et al., 2014; Ning et al., 2016; Padula et al., 2017). Currently, gel-free proteomics is the most widely used proteomics workflows; however, 2DE still has a niche in proteomics and beyond (Rabilloud, 2012; Rogowska-Wrzesinska et al., 2013; Oliveira et al., 2014; Arentz et al., 2015; Ning et al., 2016; Padula et al., 2017), as 2DE and its derivative technologies [e.g., difference gel electrophoresis (DIGE)] are highly suitable for separation of intact proteoforms (protein isoforms) and analysis of protein post-translational modifications (Tannu and Hemby, 2006; Arentz et al., 2015; Padula et al., 2017). In theory, the current 2DE technique is the same as the original method developed by O’Farrell (1975), i.e., the proteins are separated first by their isoelectric points in the first dimension of isoelectric focusing (IEF), and then by their molecular masses in the second dimension of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In practice, however, 2DE consists of several major steps, including protein extraction, IEF, after-IEF equilibration, SDS-PAGE, and protein visualization (Figure 1A). The after-IEF equilibration step is required for the reduction and alkylation of focused proteins. Since 2DE is a well-established technology, researchers working with 2DE have routinely carried out the after-IEF equilibration step without any proper understanding of its necessity. Therefore, it is time for us to rethink and optimize our approach to after-IEF equilibration in 2DE. In this article, we review the origin, protocol, and effects of after-IEF equilibration in 2DE.We also suggest incorporation of the reduction and alkylation of proteins into the sample preparation protocol before 2DE, and the omission of the after-IEF equilibration step.