What is isoelectric focusing?
Isoelectric focusing is a technique for separating different molecules by their electric charge differences with the help of carrier ampholytes. Usually performed in a polyacrylamide gel, that takes advantage of the fact that a molecule’s charge changes with the pH of its surroundings. A protein that is in a pH region below its isoelectric point (pI) will be positively charged and so will migrate towards the cathode. As it migrates through a gradient of increasing pH, however, the protein’s overall charge will decrease until the protein reaches the pH region that corresponds to its pI. At this point it has no net charge and so migration ceases (as there is no electrical attraction towards either electrode). As a result, the proteins become focused into sharp stationary bands with each protein positioned at a point in the pH gradient corresponding to its pI. The technique is capable of extremely high resolution with proteins differing by a single charge being fractionated into separate bands.