How does pH affect the overall charge of a protein in electrophoresis?

In electrophoresis, the pH of the buffer solution determines the net charge of a protein by influencing the protonation state of its acidic and basic side chains.12

Proteins are amphoteric molecules, meaning they can act as either an acid or a base depending on the environment. The relationship between pH and charge is defined by the protein's isoelectric point (pI), which is the specific pH at which the molecule carries a net zero charge—having an equal number of positive and negative charges.23

• At pH values below the pI: The protein becomes more positively charged as functional groups gain protons. Consequently, the protein migrates toward the cathode (the negative terminal).13
• At pH values above the pI: The protein becomes more negatively charged as functional groups lose protons. The protein then migrates toward the anode (the positive terminal).13
• At the pI: The protein has no net charge and does not migrate in an electric field.12

This principle allows researchers to separate proteins based on their unique charge properties.4