Molecules are distributed over a medium that has a pH gradient (usually created by aliphatic ampholytes). An electric current is passed through the medium, creating a "positive" and "negative" end. Negatively charged particles migrate through the pH gradient toward the "positive" end while positively charged particles move toward the "negative" end. As a particle moves into a pH that neutralizes its charge, it will stop following the current. Particles of the same initial charge will deposit (or focus) around the same place on the pH gradient.
- Alpha 1-antitrypsin where electrofocusing is used for diagnosis of the enzyme phenotype.