Isoelectric focusing is a type of separation where the solutes migrate based on a different principle. The separation occurs on a gel where a pH gradient has been created using ampholytes. Ampholytes are mixtures of amphoteric polyaminocarboxylic acids. This mixture possesses a range of pIs and a high buffering capacity at each pH. It is also used to create pH gradients.
When ampholytes undergo electrophoresis, each migrates to its region, an area that matches its pI. After the 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 the 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 are commonly polyacrylamide gels, but agarose and cellulose acetate can also be used.
