How does isoelectric focusing separate proteins according to charge?

Isoelectric focusing (IEF) is an electrophoretic technique that separates proteins based on their unique isoelectric points—the specific pH value at which a protein carries no net electrical charge and ceases to migrate in an electric field.12

The process functions through the following mechanism:

• pH Gradient Establishment: A medium, such as a slab gel or column, is prepared with a mixture of buffers known as ampholytes. When an electric field is applied, these ampholytes migrate to their respective isoelectric points, creating a stable pH gradient across the medium.12
• Protein Migration: When a protein mixture is introduced into this gradient, each protein migrates through the medium under the influence of the electric field.12
• Focusing: As a protein moves, it encounters different pH levels. It will continue to migrate until it reaches the specific location in the gradient where the pH matches its isoelectric point. At this position, the protein becomes electrically neutral and stops moving, effectively "focusing" into a sharp band.2

This method is highly effective for resolving proteins based on their specific charge residues and is often combined with other techniques, such as size-based separation, in two-dimensional gel electrophoresis.2