How does electrophoresis separate proteins?

Electrophoresis separates proteins by utilizing an electric field to induce migration through a medium, typically a porous gel matrix. The separation process generally relies on two primary principles:

• Size-based separation (Sieving): Proteins are often denatured and combined with a detergent, such as sodium dodecyl sulfate (SDS). This treatment gives the protein-detergent complexes a uniform charge density and shape, allowing them to be resolved strictly by size as they migrate through the gel matrix. Larger molecules move more slowly through the pores, while smaller molecules migrate more quickly.12
• Charge-based separation: Proteins possess specific charge residues that cause them to migrate toward the cathode or anode depending on the pH of the solution. In a technique called isoelectric focusing (IEF), proteins are separated along a pH gradient until they reach their isoelectric point—the specific pH at which a protein has no net charge and ceases to migrate.32

These methods can also be combined in a two-dimensional approach to provide high-resolution separation.2