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• Western Blotting (μWB) Analysis

In combination with polyacrylamide gel electrophoresis, western blotting (WB) is an invaluable analytical tool and an indispensable technique in biological sciences. It continues to serve as a powerful diagnostic approach in medical laboratories. Nevertheless, its procedure refers to multiple steps that are frequently time and resource intensive in addition to low throughput.

Nowadays, using advances in microfluidics, a microfluidic western blotting (μWB) technology has been developed to significantly optimize resources and reduce experimental cycles. More recent advancements also enable multiplexing to promote probing of various proteins. As a reliable and experienced supplier in the microfluidic project, Creative Biolabs provides rapid, sensitive, and specific μWB services to conduct the measurements of proteins to RNA to biomolecular interactions. In contrast with conventional slab-gel systems, the microfluidic design allows smaller sample volumes, faster assay times, and easier integration with automation operations.

The Principle of μWB

The WB offers qualitative information on specific proteins in a biological sample. There’re several steps in the process including separation of the proteins on a gel matrix, transfer to a membrane, and probing of the membrane with antibodies specifically binding to the protein of interest. Given the current insights afford high-throughput techniques, it would be extremely beneficial that a rapid, miniaturized, and automated WB system can be developed to validate the findings from high-throughput proteomic techniques and otherwise. One approach involves a combination of traditional polyacrylamide gel electrophoresis, transfer of isolated proteins onto membranes followed by microfluidic manipulation to facilitate the detection of different proteins. A more recent approach combines all steps in a single platform. To be specific, μWB is constructed by isotachophoretic sample stacking during sample injection, molecular weight-based separation of denatured protein solutions via the commonly used SDS-PAGE, and in situ immunoblotting with fluorescently-labeled primary and secondary antibodies.

Fig. 1 Schematic representation of the WB procedure.¹

Case Report

To validate the microfluidic chip-based assay, μWB is used to analyze the human sera (HIV immunoreactivity) and cell lysate (NFκB). Analytical performance is improved, including a short duration of 10~60 min, multiple analyte detection, mass sensitivity at the femtogram level, high-sensitivity limits (50-pM), and quantitation capability over a 3.6-log scope. The multistep assay design depends on a photo patternable and photoreactive polyacrylamide gel. The hydrophilic polymer establishes a separation matrix for protein sizing and, after transient light exposure, a protein immobilization scaffold for succeeding antibody probing of immobilized protein bands. The protein capture efficiency exceeds 75% upon sizing conditions. This microfluidic design supports the presentation of a 48-plex μWB in a standard microscope chip form. Taken together, μWB lays a foundation for rapid, targeted proteomics by merging novel specificity with throughput superiorities of multiplexing, as is related to a wide range of biological inquiry.

Fig. 2 Microfluidic western blotting.²

Services

The precision and control from microfluidic integration and photoresponsive materials accomplish progresses unprecedentedly, including the development of 48 concurrent μWB on a standard microscope slide footprint, multiplexed analysis of 3 protein targets per blot, and quantitation over a linear dynamic range of 3.6 logs with 50-pM lower limits of detection. Besides, μWB can be applied in multiplexed protein analyses of complex proteinaceous lysates, for example, crude cell sample and crude human sera. As a well-recognized specialist in the microfluidic market, Creative Biolabs is dedicated to the latest microfluidic chip development and considers that purely microfluidic technology is viable to imbuing core analytical tools with automation, quantitative capability. Furthermore, paralleling advancements have positioned protein microarrays for high-throughput proteomics duty. To be noticed, our μWB technical services have the following advantages.

• One-stop customized services for μWB microfluidic chips to detect multiple proteins;
• Scalable and automated μWB can unite protein sizing and antibody probing in a single microfluidic system;
• Experienced engineers with abundant expertise in microfluidic solutions and chip development.

At Creative Biolabs, our experts have developed an extraordinary microscale WB method based on this mechanism, which combines the internal molecular weight marker, loading control, and antibody titration in the same protocol. In contrast with the conventional methods only detecting one protein, μWB is capable of analyzing at least dozens of proteins simultaneously from a single sample, and it needs merely approximately 1% of the amount of antibody used in regular WB operation. To this end, we provide customized microfluidic chips for μWB assays conducted in a single microchannel, and our unique μWB program is comprised of multiple steps as below.

If you’d like to know more about our high-quality microfluidic chips, please directly contact us or send us an e-mail with specific requests.

References

1. Mishra, Tiwari, et al. " Protein purification and analysis: next generation Western blotting techniques." Expert Rev Proteomics. 14.11 (2017): 1037-1053.
2. Gerver, Herr, et al. " Microfluidic Western Blotting of Low-Molecular-Mass Proteins." Anal Chem. 86.21 (2014): 10625-10632.

For Research Use Only. Not For Clinical Use.