Chapter 2 - Protein Profile Comparisons of Microorganisms, Cells and Tissues using 2D Gels

Abstract

This chapter provides an overview of the current status of 2D PAGE technology for proteome analysis of microorganisms, cells and tissues, with special emphasis on 2D PAGE with immobilized pH gradients (IPGs). Although recent progress has been made in the development of alternative methods of protein separation for proteomics, there is still no generally applicable method that can replace 2D PAGE in its ability to simultaneously separate and display several thousands of proteins from complex samples such as microorganisms, cells and tissues. In particular, the development of basic IPGs up to pH 12 has facilitated the analysis of very alkaline proteins; the introduction of overlapping narrow-range IPGs permits higher resolution separations and the analysis of less abundant proteins; and the availability of ready-made IPG strips and integrated running devices such as the IPGphor have contributed towards the goal of automation. The challenges of 2D PAGE for proteome analysis, as well as the current protocol of IPG-Dalt, in particular the merits and limits of different methods for sample solubilization and sample application with respect to the pH interval used for IPG-IEF are critically analyzed, and 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 and extended separation distances for optimum resolution and detection of minor components are given.