If three proteins of 20 kDa, 60 kDa, and 120 kDa are loaded in separate lanes on an SDS-PAGE gel under identical conditions, what band pattern would you predict from top (near well) to bottom of the gel?

• A. All three will co-migrate to the same position in the gel because SDS equalizes charge-to-mass ratio so that every protein experiences identical electrophoretic force regardless of size.
• B. Top: 20 kDa, middle: 60 kDa, bottom: 120 kDa, because SDS reverses the native charge of proteins causing smaller proteins to be retained near the wells while larger ones travel faster.
• C. Top: 120 kDa, middle: 60 kDa, bottom: 20 kDa, because larger proteins migrate more slowly through the polyacrylamide matrix while smaller proteins travel farther.
• D. The pattern cannot be predicted without knowing the pH of the running buffer since SDS-PAGE separation is dominated by isoelectric focusing which requires pH information to determine migration order.

Answer: (C)