What is the purpose of ampholytes in isoelectric focusing?

• A. Maintain the polyacrylamide gel in a solid state
• B. Maintain the protein sample in a charged state
• C. Maintain the pH of the buffer solution
• D. Establish a pH gradient in the gel

Answer: (D)

The main idea here is that ampholytes generate the pH gradient used in isoelectric focusing. These small molecules have a range of pI values, and under an electric field they distribute into distinct pH zones along the gel. As the gradient forms, proteins migrate until they reach a region where the pH equals their own isoelectric point, at which point their net charge is zero and they focus there. This gradient formation and maintenance is what enables separation by pI.

Ampholytes don’t serve to keep the gel solid, nor do they simply keep the protein sample charged or fix the external buffer pH. Their essential role is to establish and stabilize the pH gradient within the gel, creating the environment where proteins can focus at their specific isoelectric points.