Practice question

Isoelectric focusing separates macromolecules based on differences in isoelectric point rather than solely molecular weight or total charge at fixed pH. A stable pH gradient is created using carrier ampholytes or immobilines immobilized in gel matrix. Proteins migrate under electric field until reaching zone where local pH equals pI, resulting in zero net charge and cessation of movement. This focusing concentrates proteins into extremely narrow bands at characteristic pH values. Distinction from SDS-PAGE which separates by size, or ion-exchange which separates by constant charge, underlies its ability to resolve isoforms differing by single charged modification.