Two-dimensional electrophoresis of proteins has preceded, and accompanied, the birth of
proteomics. Although it is no longer the only experimental scheme used in modern
proteomics, it still has distinct features and advantages. The purpose of this tutorial paper is
to guide the reader through the history of the field, then through the main steps of the
process, from sample preparation to in-gel detection of proteins, commenting the
constraints and caveats of the technique. Then the limitations and positive features of
two-dimensional electrophoresis are discussed (e.g. its unique ability to separate complete
proteins and its easy interfacing with immunoblotting techniques), so that the optimal type
of applications of this technique in current and future proteomics can be perceived. This is
illustrated by a detailed example taken from the literature and commented in detail. This
Tutorial is part of the International Proteomics Tutorial Programme (IPTP 2).