Proteomics, the analysis of the complete complement of proteins in a cell, tissue, or organism (the proteome), involves the detection of the presence or absence of proteins and the direct measurement of relative protein abundances. One of the greatest challenges of proteome analysis is the reproducible fractionation of complex protein mixtures while retaining the qualitative and quantitative relationships among component proteins. Currently, two-dimensional polyacrylamide gel electrophoresis (2-D PAGE), which is capable of resolving thousands of proteins in a single run, is the primary tool of proteomics research. This section describes the various steps of a typical 2-D electrophoresis workflow, including
A special section, the 2-D Doctor™, is a practical resource for 2-D gel troubleshooting.
Proteome analysis is used to determine which proteins in a cell, tissue, or organism are affected by changes in conditions such as disease states or developmental stages. Protein profiles in different states of a cell or an organism can be compared to identify proteins that are qualitatively and quantitatively affected by the condition of interest. Such profiling requires superior protein separation resolution and high-throughput technologies to address the potentially large numbers of proteins.
2-D electrophoresis can be used to resolve complex mixtures of thousands of proteins. In the first dimension, proteins are separated based on differences in isoelectric point (pI). In the second dimension, they are separated according to molecular weight. Following separation, 2-D electrophoresis gels are stained for protein visualization and analysis. In combination with computer-assisted image evaluation systems for comprehensive qualitative and quantitative examination of proteomes, this electrophoresis technique allows cataloging of proteins and comparison of data among groups of researchers.
The 2-D electrophoresis workflow.
The general workflow of a 2-D gel-based proteomics experiment is outlined below, and some of the factors affecting the way the experiment is performed are discussed. Bio-Rad’s 2-D Electrophoresis Workflow system provides a comprehensive set of product solutions and educational resources to help you achieve better 2-D results.
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