The advancements in two-dimensional electrophoresis (2DE) have consolidated it as a key tool for gel-based proteomics applications. Nowadays, 2DE is extensively applied and it is a useful technique for the simultaneous separation of hundreds to thousands of proteins, analysis, and differential quantification especially for the study of nonmodel organisms. This chapter focuses on all the steps involved in standard 2DE in protein expression. The pros and cons of the technique are discussed as well as potential variants for special applications. Special attention is given to data pre-processing and statistical analysis of 2DE generated data in order to draw sound conclusions from biological studies.
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