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.
How to perform Western Blotting (μWB) on microfluidic chip?
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 Western blot workflow.Distributed under CC BY-SA 4.0, from Wiki, without modification.
Case Study
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.
Services Creative Biolabs Offer
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.
Features and Benefits
Miniaturized System
The miniaturized system of μWB reduces sample and reagent consumption, making the process more cost-effective. This efficiency allows researchers to perform multiple experiments with limited sample volumes, conserving valuable resources and reducing overall costs.
Automated Workflow
Automation in μWB streamlines the Western blotting process, reducing manual intervention and the potential for human error. This leads to more consistent and reproducible results, improving the reliability of protein analysis and accelerating experimental workflows.
High Throughput Capability
μWB enables the simultaneous analysis of multiple samples, increasing throughput and efficiency. This high-throughput capability is essential for large-scale studies and screening projects, significantly speeding up the pace of research and discovery.
Faster Processing Times
The microfluidic design of μWB allows for rapid protein separation and detection, shortening the overall processing time. This quick turnaround is advantageous for time-sensitive projects, enabling faster data acquisition and decision-making.
Enhanced Sensitivity
μWB technology offers enhanced sensitivity, allowing for the detection of low-abundance proteins that might be missed with traditional Western blotting. This increased sensitivity ensures more comprehensive and accurate protein analysis, crucial for detailed studies.
Published Data
The findings displayed in the articles related to western blotting (μWB) using microfluidic chips are presented here:
1. Single-cell western blotting of chemically fixed cancer cells
Fig 2. Schematic of a hybrid microfluidic platform (DropBlot).1
Existing protein morphology analysis tools are inadequate, mainly in terms of specificity and sensitivity. Yang Liu et al. developed a hybrid microfluidic platform specifically for protein morphology analysis of chemically fixed single cells, also known as DropBlot.1 This microfluidic chip integrates two functions into one chip. The two functions are single fixed cell sample preparation from droplets and single cell protein immunoblotting. The design of DropBlot consists of three typical independent microfluidic modes, namely cell droplet, microwell and planar chip separation, thus unifying the different chemical, thermal, electrical and mechanical conditions required for each stage of the preparation and analysis process into an integrated workflow.
Reference
Liu, Yang, and Amy E. Herr. "DropBlot: single-cell western blotting of chemically fixed cancer cells." Nature Communications 15.1 (2024): 5888. Distributed under Open Access license CC BY 4.0, without modification.
Q&As
Q: How does microfluidic Western blotting (μWB) differ from traditional Western blotting?
A: μWB uses microfluidic devices to miniaturize and automate the Western blotting process. This reduces sample and reagent consumption, shortens processing time, and increases throughput compared to traditional methods. It also allows for more precise control and reproducibility of experimental conditions.
Q: What are the advantages of using μWB over traditional Western blotting?
A: Advantages include reduced sample and reagent consumption, faster processing times, higher throughput, and greater reproducibility. μWB also offers enhanced sensitivity and precision, making it a more efficient and reliable method for protein analysis.
Q: What types of samples can be analyzed using μWB?
A: μWB can analyze a variety of samples, including cell lysates, tissue extracts, and biofluids such as blood and serum. The technology is compatible with both crude and purified protein samples, making it versatile for different research needs.
Q: How is data from μWB analyzed?
A: Data analysis involves detecting and quantifying specific protein bands using advanced imaging systems and software. The results are interpreted to determine protein expression levels, post-translational modifications, and other relevant protein characteristics, providing detailed insights into the sample's proteomic profile.
Q: Can μWB be customized for specific research needs?
A: Yes, μWB can be customized to include different types of proteins, detection methods, and experimental conditions. This flexibility ensures that the technology can be tailored to specific research questions and objectives, providing highly relevant and specific data.
Resources
Videos
Creative Biolabs has established a comprehensive microfluidics technique platform.
Fig. 1 Western blot workflow.
Fig 2. Schematic of a hybrid microfluidic platform (DropBlot).1
