Which technique separates proteins by migration in a pH gradient?

• A. Endosmosis
• B. Iontophoresis
• C. Isoelectric focusing
• D. Capillary electrophoresis

Answer: (C)

The technique being tested relies on separating proteins by their isoelectric point using a pH gradient. Proteins carry different net charges depending on the pH of their surroundings. When placed in an electric field within a pH-gradient medium, they migrate toward the direction that matches their charge. As they move through the gradient, the local pH changes their charge; each protein continues until it reaches the region where the pH equals its isoelectric point, at which point it has no net charge and stops migrating. This creates narrow, focused bands—high resolution that distinguishes proteins with different pI values, including isoforms.

Other methods listed don’t use a pH gradient to halt movement at a specific point determined by the protein’s charge properties. Endosmosis involves solvent flow through membranes, iontophoresis moves charged species with current through tissues, and capillary electrophoresis separates mainly by electrophoretic mobility (charge-to-size) rather than focusing at their pI in a pH gradient.