The current state of two-dimensional electrophoresis with immobilized pH gradients

TLDR

The protocol for two‑dimensional electrophoresis with immobilized pH gradients (IPG‑Dalt) has been updated. The study provides guidelines for running conditions of analytical and micropreparative IPG‑Dalt, including use of wide IPGs up to pH 12 for overview patterns and narrow IPGs for high‑resolution zoom‑in gels. The authors critically discuss sample solubilization and application methods (cup‑loading versus in‑gel rehydration) relative to the pH interval for IPG‑isoelectric focusing, provide running‑condition guidelines, and include a brief troubleshooting guide. The paper demonstrates extended separation distances and automated procedures, and compares protein detection by silver staining with fluorescent dyes.

Abstract

The original protocol of two-dimensional electrophoresis with immobilized pH gradient (IPG-Dalt; Görg et al., Electrophoresis 1988, 9, 531—546) is updated. Merits and limits of different methods for sample solubilization, sample application (by cup-loading or in-gel rehydration) with respect to the pH interval used for IPG-isoelectric focusing are critically discussed. Guidelines for running conditions of analytical and micropreparative IPG-Dalt, using wide IPGs up to pH 12 for overview patterns, or narrow IPGs for zoom-in gels for optimum resolution and detection of minor components, are stated. Results with extended separation distances as well as automated procedures are demonstrated, and a comparison between protein detection by silver staining and fluorescent dyes is given. A brief trouble shooting guide is also included.