Protein Protocols Protein Protocols

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Difference Gel Electrophoresis
Mustafa Ünlü and Jonathan Minden
1. Introduction
Proteomics is a relatively newly coined term. It refers to the field of study of the
proteome, which was defined for the first time in 1994 as “the PROTEin products of a
genOME” (1). Part of the attractiveness of proteomics derives from its promise to
uncover changes in global protein expression accompanying many biologically relevant
processes, such as development, tumourigenesis, and so forth. Because proteins are the
effector molecules that carry out most cellular functions, studying proteins directly has
clear advantages over and (at least in theory) achieves results that go beyond genomic
analyses. Whereas the information contained in the genome is almost always static,
spatiotemporal patterns of protein expression are very complex and dynamic due to
fluctuations in abundance. This complexity is increased several-fold by proteins’ ability
to be modified functionally through posttranslational modifications.
To detect these changes in global protein expression, there is a clear requirement
that the methodology employed be able to generate and compare snapshots of the entire
protein component of any organism, cell, or tissue type. Hence, as the method that
offers the highest practical resolution in protein fractionation, two-dimensional gel electrophoresis
(2-DE) is the most commonly used separation technique in proteomics. Its
resolution power derives from orthogonally combining two separations based on two
independent parameters: isoelectric focussing (IEF) separates based on charge and
sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) separates by
size. With 2-DE, each proteome snapshot becomes a protein map, a two-dimensional
gel on which hundreds to a few thousand proteins are fractionated and displayed. Comparison
of two or more gels then allows for the detection of the protein species differing
between the compared states of the organism, cell, or tissue type under investigation.
2-DE was first described simultaneously by several groups in 1975 (2–4). Despite
the quite substantial advances in technology since its launch, the most notable of these
being the introduction of immobilized gradients in the first dimension (5), some of
the more significant systemic shortcomings have remained unsolved. The most troublesome
of these is the inherent lack of reproducibility between gels, despite the fact that
immobilized IEF gradients have alleviated this problem to some extent. Efforts to
surmount this limitation have focused on developing methods that have increasingly
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
185