Western Blotting Using Microchip Electrophoresis Interfaced to a Protein Capture Membrane

Abstract

Western blotting is a commonly used assay for proteins. Despite the utility of the method, it is also characterized by long analysis times, manual operation, and lack of established miniaturized counterpart. We report a new way to Western blot which addresses these limitations. In the method, sodium dodecyl sulfate (SDS)-protein complexes are separated by sieving electrophoresis in a microfluidic device or chip. The chip is interfaced to a moving membrane so that proteins are captured in discrete zones as they migrate from the chip. Separations of SDS-protein complexes in the molecular weight range of 11 to 155 kDa were completed in 2 min with 4 × 104 theoretical plates at 460 V/cm. Migration time and peak area relative standard deviations were 3–6% and 0.2% respectively. Detection limit for actin was 0.7 nM. Assays for actin, AMP-kinase, carbonic anhydrase, and lysozyme are shown to demonstrate versatility of the method. Total analysis time including immunoassay was 22–32 min for a single sample. Because processing membrane for immunoassay is the slow step of the assay, sequential injections from different reservoirs on the chip and capture in different tracks on the same membrane allow increased throughput. As a demonstration, 9 injections were collected on one membrane and analyzed in 43 min (~5 min/sample). Further improvements in throughput are possible with more reservoirs or parallel channels.

Applications of microfluidics and microchip electrophoresis for potential clinical biomarker analysis

“…In western blot analysis, membrane processing is a slow step that may take at least one hour. An integrated microchip electrophoresis system with western blotting developed by Jin et al [48] reduced the time to 22-32 min for a complete immunoassay. Figure 8A shows a schematic of the microchip electrophoresis device interfaced with a blotting membrane.…”

“…(B) Separation and blotting results of ladder, actin, carbonic anhydrase and lysozyme. Adapted with permission from reference [48]. …”

New developments in Western blot technology

“…In another approach, Jin et al extended the approach of using hybrid CE-protein capturing membrane apparatus by using a microfabricated glass chip for protein sieving separation [19]. Relative to the CE system, the use of a microfluidic system greatly improves the speed of separation, automates injection, and improves reliability and performance of interface to membrane.…”

Recent advances in the analysis of therapeutic proteins by capillary and microchip electrophoresis

“…The separation of a series of test proteins with a MW range of 11–155 kDa (Table 2) was performed on-chip by Jin et al [140]. Following the separation, the sample was eluted from the chip onto the Western blot membrane.…”