Two‐Dimensional Gel Electrophoresis Using the O'Farrell System

Abstract

AbstractTwo‐dimensional gel electrophoresis is the combination of two high‐resolution electrophoretic procedures (isoelectric focusing and SDS‐polyacrylamide gel electrophoresis) to provide much greater resolution than either procedure alone. In the first‐dimension gel, solubilized proteins are separated according to their isoelectric point (pI) by isoelectric focusing. This gel is then applied to the top of an SDS‐slab gel and electrophoresed. The proteins in the first‐dimension gel migrate into the second‐dimension gel where they are separated on the basis of their molecular weight. The basic protocols in this unit are based on the type of equipment originally described by O'Farrell in 1975. Such equipment is relatively inexpensive and can produce satisfactory results in situations that do not require running large numbers of two‐dimensional gels. Many variations of the original procedure have been described in the literature and by equipment suppliers. The procedures in this unit are appropriate, with minor modifications, for different equipment available from commercial suppliers or custom‐made equipment similar to that described by O'Farrell; detailed instructions are usually supplied by the manufacturer. For very basic or very acidic proteins, two alternate protocols are provided. A third alternate protocol describes how two‐dimensional electrophoresis can be performed using a minigel system. Protein sample preparation is presented in the Support Protocol.