Native and sodium dodecyl sulfate‐capillary gel electrophoresis of proteins on a single microchip

Abstract

Simultaneous electrophoresis of both native and Sodium dodecyl sulfate (SDS) proteins was observed on a single microchip within 20 min. The capillary array prevented lateral diffusion of SDS components and avoided cross contamination of native protein samples. The planar sputtered electrode format provided a more uniform distribution of separation voltage into each of the 36 parallel microchannel capillaries than platinum wire electrodes commonly used in conventional electrophoresis. The customized geometry of the stacking capillary machined into the cover plate of the microchip facilitated reproducible sample injection without the requirement for stacking gel. Polyimide served as a mask and facilitated insulation of the anode and cathode to prevent electrode lift off and deterioration during continuous electrophoresis, even at a constant current of 8 mA. Improved protein separation was observed during capillary electrophoresis at lower currents. Ferguson plot analysis confirmed the electrophoretic mobility of native globular proteins in accordance with their charge and size. Corresponding Ferguson plot analysis of SDS-associated proteins on the same chip confirmed separation of marker proteins according to their molecular weight.

SDS‐CGE of proteins in microchannels made of SU‐8 films

“…Migration time of proteins at a distance of 2.5 cm vs. the molecular weight for four different polyacrylamide gels (6,8,10, and 12%). Tsai et al [8] reported Ferguson plots of four proteins with molecular weights between 32 and 85 kDa. The experiments were carried out in glass channels.…”

“…Yao et al [2] successfully separated six fluorescently labelled proteins ranging from 9 to 116 kDa in a glass microchannel, in less than 35 s. Tsai et al [8] also reported a simultaneous CGE of both native and SDS proteins in a glass device within 20 min.…”

Intact-protein-based High-resolution Three-dimensional Quantitative Analysis System for Proteome Profiling of Biological Fluids

“…For example, ion exchangebased separation may be substituted for ampholyte-based IEF in the first dimension, and size-based separation in the third dimension may be accomplished by capillary electrophoresis using polymers as a molecular sieve matrix that allows proteins to be resolved based on molecular mass (20 -22). Capillary electrophoresis in the third-dimension separation could be multiplexed to increase throughput and would facilitate protein recovery by eliminating the need for gel cutting and protein extraction from gels (23)(24)(25). Automation of the separation system can also be envisaged.…”

Advanced Capillary and Microchip Electrophoretic Techniques for Proteomics

“…method provided the LODs of 0.1 ng/µL for fluorescently labeled proteins. A simultaneous electrophoretic separation of both native and denatured proteins (SDS-proteins) in 36 parallel microchannels on a single microchip within 20 min was demonstrated (Tsai et al, 2004). The versatile microchannel array with self-contained electrodes allows Ferguson plots of native and denatured proteins obtained and provides new opportunities for analysis of many different protein samples by a single separation run.…”