protein separation by isoelectric focusing

Meaning: The gel holds a stable pH gradient, acidic at the anode (+) and basic at the cathode (−). A protein's net charge depends on the pH around it. Where pH is below its pI, the protein is net positive and moves toward the cathode. Where pH is above its pI, it is net negative and moves toward the anode. It stops at the position where pH = pI, because net charge is then zero and the field no longer moves it. If it drifts away, it regains charge and is pushed back, so the bands are sharp.

Equation: net charge = 0 when pH = pI. For an amino acid with no ionizable side chain, pI = (pKa1 + pKa2)/2, where pKa1 and pKa2 are the acid dissociation constants of the carboxyl and amino groups (unitless).

On the MCAT: You may be given pI values and asked which way a protein moves at a stated pH, or where it ends up in the gradient. A protein rich in Asp and Glu has a low pI. One rich in Lys, Arg and His has a high pI. Passages may also compare IEF with SDS-PAGE, which separates by mass, or describe 2D gels. In a 2D gel, IEF usually comes first and SDS-PAGE second.

Common confusion: Students treat IEF like SDS-PAGE, where proteins keep running toward one electrode and separate by size. In IEF, migration stops at pH = pI, and charge, not size, determines the final position.