Practice question

Discontinuous electrophoresis depends on Tris-glycine discontinuous buffer system to create transient isotachophoretic stacking. Chloride ions from Tris-HCl serve as highly mobile leading ions. Glycine is amphoteric with pKa2 near 9.6; at stacking gel pH 6.8 it exists predominantly as zwitterion with very low electrophoretic mobility, acting as trailing ion, while proteins possess intermediate mobility. Resulting Kohlrausch boundary generates steep voltage gradient that focuses proteins into micrometer-thin zones. APS and TEMED drive radical polymerization, and acrylamide forms sieving matrix; they do not establish pH or mobility gradient essential for concentration effect.