The principle of isoelectric focusing. A pH gradient is established in a gel before loading the sample. (A) Each protein, represented by the different colored circles, will possess a net positive charge in the regions of the gel where the pH is lower than its respective pI value and a net negative charge where the pH is greater than its pI. When voltage is applied to the gel, each protein will migrate to its pI, the location at which it has no net charge. (B) The proteins form bands that can be excised and used for further experimentation.