Protein separation using preparative‐scale dynamic field gradient focusing

Abstract

Dynamic field gradient focusing uses an electric field gradient to separate and concentrate proteins in native buffers. A prototype preparative-scale dynamic field gradient focusing apparatus reproducibly separated hemoglobin and bovine serum albumin with a mean resolution of 2.64 ± 0.503. Run-to-run variations in the hemoglobin’s focal point and peak width appeared to be related to fluctuations in the shape of the electric field, rather than the 5% accuracy of the pump that provided the counter-flow in the separation annulus. The variation in the electric field gradient was probably due to the formation and expansion of an ion-depleted region at the top of the separation annulus.

Direct current dielectrophoretic simulation of proteins using an array of circular insulating posts

“…Dielectrophoresis (DEP), a non-invasive and non-destructive technique, is employed in lab-on-a-chip (LOC) platforms to manipulate bioparticles such as cells [1][2][3][4][5], proteins [6,7], DNA [8][9][10][11], viruses [12][13][14][15], and bacteria [16,17]. It is a phenomenon in which, a force is exerted on a dielectric particle when subjected to a non-uniform electric field [1, 18,19].…”

Counterflow gradient electrophoresis for focusing and elution

“…For an additional DFGF apparatus, Tracy and Ivory were interested in increasing the amount of separated analyte so DFGF could be used at the preparative scale . With an 18 mL chamber, Tracy et al separated 7 mg of each of two different proteins . To minimize axial dispersion in such a large chamber, a rotor was used to turn the chamber and induce vortices, which led to radial‐dominated dispersion.…”

Influence of the semi‐permeable membrane on the performance of dynamic field gradient focusing

“…More recently, Tracy and Ivory [11, 28–30] published a series of papers describing the development of a preparative scale DFGF apparatus capable of processing tens to hundreds of milligrams of protein. Unlike previous DFGF designs, Tracy and Ivory were able to mitigate the need for a stationary phase by basing their design on that of a vortex-stabilized electrophoresis apparatus [31].…”

“…Unlike previous DFGF designs, Tracy and Ivory were able to mitigate the need for a stationary phase by basing their design on that of a vortex-stabilized electrophoresis apparatus [31]. In this case, an annulus is turned within a stationary cylinder, introducing counter-rotating vortex pairs that reduce axial dispersion along the length of the separation channel at the expense of radial dispersion [11]. Using this design, they were able to separate 7mg each of hemoglobin and FITC-BSA.…”