Principle of Isoelectric Focusing

The use of isoelectric focusing (IEF) is limited to molecules which can be either positively or negatively charged. Proteins, enzymes and peptides are such amphoteric molecules. The net charge of a protein is the sum of all negative and positive charges of the amino acid side chains, but the three-dimensional configuration of the protein also plays a role.

Isoelectric focusing takes place in a pH gradient. The charged molecules move towards the anode or the cathode until they reach a position in the pH gradient where their net charges are zero. This pH value is the "isoelectric point" (pI) of the substance. Since it is no longer charged, the electric field does not have any influence on it. Should the substance diffuse away, it will gain a net charge again, and the applied electric field will cause it to migrate back to its pI. This concentrating effect leads to the name focusing. Thus with IEF there is no problem with diffusion.