Isoelectric focusing is a type of separation where the solutes migrate based upon a different principle. The separation takes place on a gel where a pH gradient has been created using ampholytes. Ampholytes are a mixtures of amphoteric polyaminocarboxylic acids. This mixture possesses a range of pIs, a high buffering capacity at each pH, and is used to create pH gradients.
When ampholytes undergo electropohoresis, each individual ampholyte migrates to its own region, an area that matches its pI. After migration of ampholytes, the gel has stable pH zones of increasing pH or a pH gradient. The solutes in the specimen do not migrate to the electrode of opposite charge but to the zone or area that matches their pI.
IEF is performed on a gel in a capillary tube, strip, or plate. Gels used are most commonly polyacrylamide gels but agarose and cellulose acetate can also be used.