Isoelectric focusing as the crow flies.

The evolution of isoelectric focusing is traced back over the years, from a somewhat shaky origin to present-day immobilized pH gradients. Four generations of methodology are classified and discussed: (A) Kolin's approach, consisting of a two-step technique, generation of a pH gradient by diffusion followed by a rapid electrokinetic protein separation; (B) Svensson-Rilbe's approach, consisting of creating a pH gradient in an electric field by utilizing as buffers a multitude of carrier ampholytes, i.e. of amphoteric species possessing good buffering capacity and conductivity at their pI; (C) immobilized pH gradients, by which non-amphoteric buffers and titrants (acrylamido weak acids and bases), titrated around their pK values, are grafted (insolubilized) onto a polyacrylamide gel matrix and (D) mixed-bed carrier ampholyte-Immobiline gel, by which a soluble, carrier ampholyte generated pH gradient coexists in the same matrix with an insoluble, Immobiline generated, pH gradient.