High‐resolution computer simulation of the dynamics of isoelectric focusing of proteins

Abstract

A dynamic electrophoresis simulator that accepts 150 components and voltage gradients employed in the laboratory was used to provide a detailed description of the focusing process of proteins under conditions that were hitherto inaccessible. High-resolution focusing data of four hemoglobin variants in a convection-free medium are presented for pH 3-10 and pH 5-8 gradients formed with 20 and 40 carrier ampholytes/pH unit, respectively. With 300 V/cm, focusing is shown to occur within 5-10 min, whereas at 600 V/cm separation is predicted to be complete between 2.5 and 5 min. The time interval required for focusing of proteins is demonstrated to be dependent on the input protein charge data and, however less, on the properties of the carrier ampholytes. The simulation data reveal that the number of transient protein boundaries migrating from the two ends of the column towards the focusing positions is equal to the number of sample components. Each protein is being focused via the well-known double-peak approach to equilibrium, a process that is also characteristic for focusing of the carrier ampholytes. The predicted focusing dynamics for the hemoglobin variants in pH 3-10 and pH 5-8 gradients are shown to qualitatively agree well with experimental data obtained by whole-column optical imaging.

Capillary electrophoresis of proteins 2003–2005

“…A simulation program accepting 150 components was used to simulate dynamics of IEF of proteins [13]. Computer simulation of focusing dynamics of hemoglobin was found to be in accordance with experimental data.…”

Dynamic computer simulations of electrophoresis: A versatile research and teaching tool

“…This is illustrated here with ZE, ITP, IEF and EKC separation examples. The data shown were produced with GENTRANS, the simulator based on the work of Bier, Mosher, Thormann, Breadmore and coworkers [1,37,38,44,[49][50][51][52][53][54]75].…”

“…With the significant improvement in computer power over the last twenty years, the capacity of GENTRANS could be increased to handle up to 150 components and to perform high-resolution IEF simulations at experimental current densities [53,54,132,159,160,177]. Mao et al [53] presented simulation data of a pH gradient spanning seven pH units from 3-10, with the pH gradient made from 140 Electrophoresis 2010, 31, 726-754 ampholytes, and low molecular mass amphoteric dyes as sample constituents.…”

“…The data suggest that it is impossible to reach a completely stationary steady-state pH gradient when using carrier ampholyte solutions, and that the only way to achieve this is through the use of immobilized pH gradients such as those of Immobiline gels. Thormann et al [54] performed simulations of focusing of hemoglobin variants and compared these with whole column imaging IEF (both approaches at 300 and 600 V/cm). They simulated a broad range pH gradient from 3-10 with 20 ampholytes per pH unit (140 ampholytes over the entire gradient) and a narrow range pH gradient from 5-8 with 40 ampholytes per pH unit.…”

“…New models with sophisticated integration schemes emerged and the number of applications increased. Models that are currently in use in various laboratories are the 1-D simulators GENTRANS of Mosher et al [44,49,[51][52][53][54][129][130][131][132] and SIMUL5 of Hruška et al [48] (available at http://www.natur.cuni.cz/ Gas), which succeeded SIMUL 4.0 [47]. Interesting new models are those of Bercovici et al [69] (available as the Stanford's Public Release Electrophoretic Separation Solver (SPRESSO), http://microfluidics.stanford.edu/spresso), the 2-D model of Shim et al [70,71,135,136] and the models of Yang and his collaborators [68,138].…”