Large molecules, such as proteins, can contain many acidic and basic groups. Such molecules are called polyampholytes. With more than two charged groups present, the calculation of pI becomes more complicated. However, as long as the molecule has both positively and negatively charged groups, it always has an isoelectric point, at which the net average charge is zero.

If acidic groups predominate, the pI will be low. If basic groups predominate, the pI will be high.