How do protein size and electric charge affect electrophoresis results?

In electrophoresis, protein separation is primarily influenced by the interplay between molecular charge and size within an electric field.12

Influence of Electric Charge

Proteins possess both positive and negative charges, and their net charge depends on the pH of the surrounding solution. In an electric field, proteins migrate toward the electrode with the opposite charge: negatively charged proteins move toward the anode, while positively charged proteins move toward the cathode. At a specific pH known as the isoelectric point, a protein has no net charge and will not migrate. This principle is utilized in isoelectric focusing to separate proteins based on their unique charge residues.231

Influence of Size

When electrophoresis is conducted in a porous gel matrix, the gel acts as a sieve. The matrix creates resistance, causing larger, heavier molecules to move more slowly, while smaller, lighter molecules migrate more quickly. To isolate size as the primary variable, proteins are often denatured and treated with a detergent like sodium dodecyl sulfate (SDS), which imparts a uniform charge density to the molecules, allowing them to be resolved strictly by size.14