Two-Dimensional PAGE Using Immobilized pH Gradients
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Since its introduction some 20 years ago by O’Farrell (1), two-dimensional polyacrylamide gel electrophoresis (2D PAGE) has remained an extremely powerful method for the separation of complex protein mixtures, such as whole-cell lysates. In the first dimension, proteins are separated by charge using isoelectric focusing (IEF) and, in the second, by size employing sodium dodecyl sulfate (SDS)-PAGE. The combination of these two techniques results in a highly resolving separation that can be used for many applications, such as gene regulation studies, comparison of protein phosphorylation, and protein purification. At higher loadings, identification of individual proteins can be carried out using such methods as microsequencing or mass spectrometry.
KeywordsSodium Dodecyl Sulfate Carrier Ampholyte Complex Protein Mixture Gene Regulation Study Plastic Backing