The Basics of 2D DIGE - Difference Gel Electrophoresis (DIGE): Methods and Protocols - page 9

Chapter 2
The Basics of 2D DIGE
Phil Beckett
Abstract
The technique of two-dimensional (2D) gel electrophoresis is a powerful tool for separating complex
mixtures of proteins, but since its inception in the mid 1970s, it acquired the stigma of being a very diffi -
cult application to master and was generally used to its best effect by experts. The introduction of com-
mercially available immobilized pH gradients in the early 1990s provided enhanced reproducibility and
easier protocols, leading to a pronounced increase in popularity of the technique. However gel-to-gel
variation was still diffi cult to control without the use of technical replicates. In the mid 1990s (at the same
time as the birth of “proteomics”), the concept of multiplexing fl uorescently labeled proteins for 2D gel
separation was realized by Jon Minden's group and has led to the ability to design experiments to virtually
eliminate gel-to-gel variation, resulting in biological replicates being used for statistical analysis with the
ability to detect very small changes in relative protein abundance. This technology is referred to as 2D
difference gel electrophoresis (2D DIGE).
Key words: Two-dimensional gel electrophoresis, 2D DIGE, CyDye, Multiplexing, Difference gel
electrophoresis
1. Introduction
Two-dimensional (2D) gel electrophoresis allows for the simulta-
neous separation of thousands of proteins and was pioneered by
O'Farrell ( 1 ), utilizing a denaturing environment. The technique
separates the proteins based on their charge in the fi rst dimension
using isoelectric focusing (IEF), where the proteins will migrate to
their isoelectric point (pI). In the second dimension, the proteins
are separated based on molecular weight by the use of classical
SDS-PAGE ( 2 ). The traditional use of carrier ampholytes to estab-
lish the pH gradient in the fi rst dimension led to a number of
issues, most notably the lack of reproducibility. This resulted in the
development of immobilized pH gradients ( 3 ), leading to com-
mercially available precast gels for the fi rst dimension that allowed
for enhanced reproducibility (since they do not rely on carrier