Isoelectric focusing – a cheap and effective process for the simultaneous concentration and separation of proteins. Heterogeneous protein samples from purified as well as full cell extracts can be separated into their constituent parts.
In horizontal isoelectric focusing an electric charge is applied to a gel soaked in a mixture of many different, small, amphoteric molecules (carrier ampholytes). This generates a pH gradient along which the proteins migrate until they reach their isoelectric point (i.e. the pH at which their overall charge is neutral).
Different proteins appear as separate zones only if zwitterionic molecules with appropriately tuned isoelectric points are available to go in between. Very similar proteins require a great multitude of spacer-molecules – Sepalyte carrier ampholytes have this polyfunctional character, which is essential for sharp separations.