In SDS-PAGE, proteins migrate primarily according to which property?

• A. Isoelectric point
• B. Size (molecular weight)
• C. Hydrophobicity
• D. Net native charge

Answer: (B)

SDS-PAGE separates proteins mainly by size. In this technique, proteins are treated with SDS, a detergent that binds along the polypeptide chain and unfold it into a extended, rod-like form while imparting a roughly uniform negative charge per unit length. Because the charge-to-mass ratio is largely constant for different proteins, their movement through the polyacrylamide gel depends primarily on how easily their linear chains can move through the gel’s pores. Smaller proteins fit through the pores more easily and migrate faster toward the positive electrode, while larger proteins slow down. Hydrophobicity and the native net charge don’t drive separation here, since SDS masking and denaturation minimize those factors. The isoelectric point is a critical factor in methods like isoelectric focusing or native PAGE, not in SDS-PAGE.