Field-Free Remobilization of Proteins after Isoelectric Focusing in Packed Capillaries

Abstract

Pressure-driven remobilization without an applied electric field is shown to be possible with capillary isoelectric focusing by using packed capillaries. The capillary dimensions are 100 μm i.d. and 2 cm in length, and the packing is made of 0.9 μm nonporous silica particles that are chemically modified with a brush layer of polyacrylamide. Both reversible and irreversible adsorption are shown to be negligible. The packed capillaries eliminate the problem of unwanted hydrodynamic flow between reservoirs. Three proteins are focused: trypsin inhibitor, carbonic anhydrase II and myoglobin. The time required for focusing in the packed capillaries is increased by only a factor of two compared to the open capillary, giving complete focusing in less than 15 min at 200 V/cm. The packed capillaries allow the use of higher electric fields, with resolution continually increasing up to at least 1500 V/cm. The packing obstructs diffusional broadening after the field is turned off: for trypsin inhibitor, D=6.1(±0.3)×10-8 cm2/s for the packed capillary vs. D=28.8(±0.3)×10-8 cm2/s for the open capillary. The broadening contributed by the packing during remobilization is from eddy diffusion, and it is described by its plate height, H, which is the variance per unit length: H=σ2/L=0.64 μm. This limits the resolution to 0.1 pH unit for the 2-cm capillary having a pH range of 3-10, giving a theoretical peak capacity of 47.

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide

“…A, which shows that the hydrated polyacrylamide occupies some of the volume, thereby reducing the pore radius to ξ. The assumption that the proteins are excluded from the polyacrylamide layer is supported by earlier work . The water inside the polyacrylamide layer is thereby taken to behave as a stagnant layer that is porous only to water and other small molecules .…”

Modeling of protein electrophoresis in silica colloidal crystals having brush layers of polyacrylamide

“…The packed capillaries were modified with polyacrylamide, as detailed previously [12,15]. Briefly, the silica surface was silylated with inititor, and linear polyacrylamide chains were grown using a complex of CuCl with tris (2-dimethylaminoethyl) amine as the catalyst.…”

“…For capillary experiments, dual reservoir PDMS holders were placed on a glass slide to prevent electrophoresis gas bubbles from entering the channel [11,12]. The capillaries packed with 350 nm and 500 nm particles were saturated with a commonly used protein electrophoresis TGS running buffer composed of 25 mM tris, 192 mM glycine, 0.1% (w/v) SDS (pH 8.0) overnight, and electrically conditioned at 50 Vcm −1 using a high-voltage power supply (10A25A Series, Ultravolt, Ronkonkoma, NY) controlled by LabView until the current became static.…”

“…A new approach is to create a rigid porous network with colloidal silica, which forms facecentered cubic crystals in thin channels [10] and in capillaries [11]. The solid matrix gives high thermal conductivity to reduce broadening due to heating [12]. Work by Zeng et al was the first to demonstrate that sieving of proteins and DNA fragments occurs in silica colloidal crystals [10].…”

Trajectory of isoelectric focusing from gels to capillaries to immobilized gradients in capillaries

“…Our own group investigated the influence of a packing material on CIEF and pressure‐driven remobilization . We used capillaries packed with nonporous 1‐μm diameter silica spheres bearing a covalently monolayer of linear polyacrylamide chains.…”