electrophoresis of proteins

Meaning: Electrophoresis separates charged molecules by their movement through a gel in an electric field. Samples are loaded into wells in a polyacrylamide gel, and the gel's pores slow larger molecules more than smaller ones. In native PAGE the folded protein migrates according to its charge, size and shape. In SDS-PAGE, the detergent SDS denatures the protein and coats it with negative charge roughly proportional to its mass, so separation is by size. Isoelectric focusing uses a pH gradient, and a protein stops where the pH equals its pI.

Equation: F = qE, where F is the electric force on the protein (N), q is its net charge (C), and E is the field strength (V/m). Drag from the gel opposes this force.

On the MCAT: Passages show a gel image with bands. Bands nearest the wells are the largest proteins, and bands that traveled farthest are the smallest. Comparing gels run with and without β-mercaptoethanol, which breaks disulfide bonds, reveals subunits held together by disulfides. Native-gel questions ask whether a protein is positive or negative at a given pH by comparing that pH with its pI.

Common confusion: Students assume SDS-PAGE separates proteins by their natural charge. SDS masks the intrinsic charge, so mass decides, and smaller proteins run farther toward the anode (the positive electrode).