Gel electrophoresis is a technique used to separate molecules based on size or charge. Charged particles can be separated because they migrate towards different ends of the gel.

Negatively charged particles always migrate towards the positive pole whereas positively charged particles always migrate towards the negative pole (opposites attract). In gel electrophoresis, the positive pole is called the anode and the negative pole is called the cathode; therefore, the charged particles will migrate to the respective nodes.

The isoelectric point is the pH at which a charged particle loses its charge and becomes neutral; therefore, the pI is only a property of the species (not its environment). When the pH of the environment equals the pI of the molecule, the electrical charge on the molecule disappears. A basic environment is characterized by a high pH. If the pH of the environment is higher than the pI, then the environment is basic. The size of the species is usually represented by mass or weight, not by pI.

pI of a substance affects other chemical properties, such as solubility. Charged species are polar and are likely to dissolve in polar solvents whereas uncharged species (when pH = pI) are likely nonpolar and are more soluble in nonpolar solvents.

A pH gradient in a gel facilitates the separation of charged species. The proteins in the mixture encounter different pHs as they travel through the gel. The driving force for the proteins is the electric force. Recall that each protein has an isoelectric point; when the protein's environmental pH equals the isoelectric point, the protein will become neutral and stop migrating (it loses the electric force). The physiological pH is about 7.4. Several amino acids are uncharged at this pH; however, these amino acids will become charged when presented in a different pH environment; therefore, these proteins will be charged and be able to migrate through the gel because of the pH gradient.

The protein will stop migrating when the pH of the environment (gel) equals the pI of the protein. Different types of gel electrophoresis are used to separate molecules based on charge and size. A pH gradient is used to separate molecules based on charge, whereas SDS-PAGE is used to separate molecules based on size. All types of macromolecules (nucleic acids, proteins, lipids, and carbohydrates) can be separated using gel electrophoresis.

SDS is a substance added to polyacrylamide gels to separate substances based on size. This method is called SDS-PAGE. SDS binds to the secondary structure of proteins and gives them an overall negative charge; the bigger the substance the bigger the negative charge. The larger mass compensates for the bigger charge; therefore, SDS makes it so that the mass-to-charge ratio of all substances is the same. This standardizes the electric force experienced by each molecule, making size the only driving force for migration through the gel.