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This handbook is intended to provide guidelines for performing high-resolution two-dimensional (2-D) electrophoresis. It is divided into seven chapters: Chapter 1 provides guidelines for sample preparation and protein quantitation. Chapter 2 details procedures for performing the first dimension of 2-D electrophoresis, highlighting use of Ettan™ IPGphor™ II Isoelectric Focusing System. Chapter 3 contains general directions for subsequent second-dimension electrophoresis of immobilized pH gradient (IPG) strips using various vertical gel electrophoresis systems. Chapter 4 describes use of the flatbed Multiphor™ II Electrophoresis System for both first- and second-dimension electrophoresis. Chapter 5 discusses visualization and analysis of 2-D electrophoresis results. Chapter 6 describes the advantages and use of the technique of 2-D Fluorescence Difference Gel Electrophoresis (2-D DIGE). Chapter 7 describes common problems in 2-D gel electrophoresis and their remedies. Technique-specific troubleshooting guides are included in the relevant chapters.