Western blotting using capillary electrophoresis

Abstract

A microscale Western blotting system based on separating sodium-dodecyl sulfate protein complexes by capillary gel electrophoresis followed by deposition onto a blotting membrane for immunoassay is described. In the system, the separation capillary is grounded through a sheath capillary to a mobile X-Y translation stage which moves a blotting membrane past the capillary outlet for protein deposition. The blotting membrane is moistened with a methanol and buffer mixture to facilitate protein adsorption. Although discrete protein zones could be detected, bands were broadened by ∼1.7-fold by transfer to membrane. A complete Western blot for lysozyme was completed in about one hour with 50 pg mass detection limit from low microgram per milliliter samples. These results demonstrate substantial reduction in time requirements and improvement in mass sensitivity compared to conventional Western blots. Western blotting using capillary electrophoresis shows promise to analyze low volume samples with reduced reagents and time, while retaining the information content of a typical Western blot.

Figure 1

Instrument overview. Sample is injected at the inlet of the separation capillary (A). The protein mixture migrates the gel-filled capillary under an electric field that is generated by the application of negative high voltage (A) and ground (D). Proteins exit the capillary as it drags over the surface, and deposit on the blotting membrane (C). A translational stage moves the blot past the end of capillary to preserve the protein separation on the membrane. Gel pumped through a sheath capillary (B) that surrounds the latter portion of the separation capillary and and makes direct contact with the blotting membrane (E). The blotting membrane (and wick overlay) are moistened with 50:50 (v:v) methanol: electrophoresis buffer.

Figure 2

Size-dependent separation of standard FITC-labeled proteins. (A) 3 proteins, prepared in stock samples of 100-300 μg/mL. The molecular weight for unlabeled protein is noted beside each observed peak. (B) Plotting log MW as a function of mobility yields a linear plot for these FITC-labeled proteins.

Figure 3

Measurements of band broadening inside sheath capillary and on membrane. (A) Comparison of peak width for on-column detection (black line), in sheath 350 μm beyond the exit of the separation capillary (dashed line), and on membrane after traveling through 500 μm of sheath (gray line). The on-column and in sheath measurements were taken from the same separation. The membrane data was from a separate injection. All separations used 150 μg/mL FITC-BSA as the sample separated at 300 V/cm with an effective capillary length of 20 cm. Capillaries were not thermostatted. (B) Selected images of protein exiting the separation capillary and entering sheath capillary. Time zero represents time zone first appears at exit of separation capillary and is 32 minutes after sample injection. The white dots indicate where signal was measured to construct Figure 3A.

Figure 4

CE-based Western blot of lysozyme at 3 different concentrations. Samples were separated at 300 V/cm and the resulting membranes probed with antibody using an automated system for applying reagents. Analysis time for an individual assay was about 60 min, though the immunoassay and detection were performed in parallel. Enlargement shows that zones spread perpendicular to the deposition track. The sheath capillary was 250 μm in diameter but the zone is 450 μm wide.

Figure 5

Immunoassay of unlabeled sample proteins at low levels. Estimated quantities of proteins calculated from injection length, elution time and sample concentrations are displayed for different proteins.

Figure 6

Immunoassay of unlabeled peak with labeled size standards. (A) Initial fluorescence scan displays FITC-labeled size standards at approximately 16 and 28 minutes for insulin (FITC-ins) and bovine serum albumin (FITC-BSA). (B) Upon immunoassaying this PVDF membrane with anti-carbonic anhydrase IgG, the intermediate MW protein, carbonic anhydrase (CA), is detected at 22 minutes.