SDS capillary gel electrophoresis of proteins in microfabricated channels

Abstract

Analysis of variations in the concentrations or structures of biomolecules (e.g., mRNAs, proteins, peptides, natural products) that occur either naturally or in response to environmental or genetic perturbations can provide important insight into complex biological processes. Many biological samples are mixtures that require a separation step before quantitation of variations in the individual components. Two-dimensional denaturing gel electrophoresis has been used very effectively to separate complex mixtures of proteins, but it is time consuming and requires considerable amounts of sample. Microchannel-based separations have proven very effective in rapidly separating small amounts of nucleic acids; more recently, isoelectric focusing of proteins also has been adapted to the microchannel format. Here, we describe microchannel-based SDS capillary gel electrophoresis of proteins and demonstrate the speed and high resolution it provides. This development is an important step toward the miniaturization and integration of multidimensional and array separation methods for complex protein mixtures.

Figure 1

Schematic of the instrumentation set up used for chip-based SDS/CGE separations. The microchannel layout used in the chip-based SDS/CGE separations also is shown. F1, excitation bandpass filter; F2, emission bandpass filter; PMT, photomultiplier tube; TS_x,y,z, x-y-z translational stage.

Figure 2

Electropherograms from capillary-based SDS/CGE on a six-protein mixture with an eCAP-coated capillary (a) and an uncoated fused-silica capillary (b). Capillary dimensions: 100-μm i.d. × 20 cm (27 cm in total length). Separation voltage: 8.1 kV. Injection: 4 kV for 30 sec. Peaks: (1) calmodulin (MM = 9 kDa); (2) α-lactalbumin (MM = 14.4 kDa); (3) pepsinogen (MM = 39 kDa); (4) egg albumin (MM = 45 kDa); (5) BSA (Mm = 66 kDa); and (6) β-galactosidase (MM = 116 kDa). Each protein concentration in the mixture was ≈1 × 10⁻⁹ M to 10 × 10⁻⁹ M. Injection: 4 kV for 30 s. (These conditions were optimized.)

Figure 3

Electropherograms of protein mixture (Fig. 2) with microchannel-based SDS/CGE with different separation voltages: 1 kV (a), 2 kV (b), 3 kV (c), 4 kV (d), and 5 kV (e).

Figure 4

Electropherograms of three cysteine-containing, fluorescently labeled proteins (peak 1, calmodulin with an apparent MM of 9 kDa; peak 2, lysozyme (hen egg white) with a MM of 14.5 kDa; peak 3, staphylococcal nuclease 16-cys mutant with a MM of 18 kDa). (a) Capillary-based SDS/CGE with the proteins labeled nonspecifically with fluorescein-NHS (Top) or labeled specifically with fluorescein-MAL (Bottom). Capillary: 100-μm i.d. × 20 cm (27 cm total length) uncoated fused-silica. Separation voltage: 8.1 kV. Injection: 4 kV for 30 s. Note the two different free dyes migrate differently under identical separation conditions. (b) Chip-based SDS/CGE of the specifically labeled proteins with different separation voltages. The Inset highlights the three well resolved protein peaks at a separation voltage of 5 kV. Each protein concentration in the mixture was estimated to be ≈1 × 10⁻⁹ M to 10 × 10⁻⁹ M.