Experimental and theoretical dynamics of isoelectric focusing: III. Transient multi-peak approach to equilibrium of proteins in simple buffers

Abstract

Transient states in the isoelectric focusing of proteins in simple three-component buffer systems were examined by (i) analyzing the collected fractions from continuous flow electrophoresis at a range of operating conditions, (ii) photographing the behavior of colored proteins in capillaries, (iii) monitoring the electric field dynamics along a capillary with a potential gradient array detector and (iv) computeer simulation. The good agreement between simulation and experimental data clearly reveals how the separation dynamics of the buffer system, i.e., the formation of a natural pH gradient, produces the observed peaks and boundaries of protein during the approach to the steady state. The protein focusing dynamics are different, but characteristic, for each buffer system, with both transient double, and multiple, peaks being observed.