Isoelectric focusing (IEF) is one of the most commonly used techniques for the separation of proteins. IEF separations are based on the pH dependence of the electrophoretic mobilities of the protein molecules. Isoelectric focusing makes use of electrical charge properties of molecules to focus them in defined zones in a separation medium. It is the focusing mechanism that distinguishes IEF from other separation processes and makes it unique among the separation methods. In sharp contrast, the basic separation mechanism of IEF imposes forces on the molecules that directly counteract the dispersive effects of diffusion. During the separation process, the molecules in the sample accumulate in specific and predictable locations in the medium, regardless of their initial distribution. This focusing mechanism also distinguishes IEF from various modes of electrophoresis. It is important to note that IEF is a high-resolution method. It is well suited for both analytical and preparative applications.
GENERATION OF pH GRADIENTS
IEF is a very useful practical technique where simple methods for establishing and maintaining pH gradients are available. The pH gradient is essential for this technique, and the nature of the pH gradient largely determines the quality and usefulness of the separation. The two most widely adopted methods for generating pH gradients make use of different types of synthetic buffering molecules (a) Carrier ampholytes are amphoteric electrolytes that carry both current and buffering capacity. They possess both acidic and basic functional groups and form pH gradients under the influence of electric fields. (b) Synthetic buffer compounds containing reactive double bonds, called acrylamide buffers, can be incorporated into polyacrylamide gel matrices. When used in the correct proportions, acryl- amido buffers generate immobilized pH gradients under the influence of electric fields.
PROTEIN DETECTION AND IMAGING
Gel-based IEF has a strong worldwide user base that supports a commercial pipeline of instrumentation as well as consumable products and will thus certainly remain a relatively low-cost, routine laboratory technique in the coming years. These techniques involve the use of many of the same dyes and stains that were developed for polyacrylamide electrophoresis gels but have been adapted for use with IEF gels.