Coexistence of steady state and transient state in isoelectric focusing

The possibility of transforming a wide (pH 3.5-10) into a narrow pH gradient in isoelectric focusing (IEF) has been examined by the use of different amino acid terminators at the electrons, viz 50 mM Asp, 50 mM Trp, 45 mM Phe. 0.5 M Gly or 0.5 M Lys. The ph gradient formation and decay seem to be insensitive to the type of amino acid used or to its molarity. While Ampholine and protein samples reach equilibrium positions, large amounts of amino acids move as two wave fronts, from the anode and cathode, unsuccessfully trying to reach their pI positions. Thus, the steady state and transient state coexist in IEF and are quite insensitive to each other. The formation and stabilization of the pH gradient in IEF do not require a performed "pH-cage", nor can IEF be regarded as isotachophoresis, since the same ion acts simultaneously as the "leading" and "terminating" ion in our system. When the same buffer is used simultaneously at the anode and cathode, the gel loses its polarity so that the system becomes insensitive to the choice of electrode position, or to polarity reversal at any time during the IEF experiment.