Muhammad Uses Simple Western to Chart Signaling Pathways in Stem Cells

Studying cell signaling for regenerative medicine

Signaling pathways regulate many central functions of living cells and understanding their mechanism of action could lead to breakthroughs in regenerative medicine. Dr. Muhammad Dain Yazid, a scientist at the Center for Tissue Engineering and Regenerative Medicine in Malaysia, uses stem cells as a model to study how growth factors and bioactive compounds induce signaling pathways in cellular processes like the endothelial-to-mesenchymal transition. Ultimately, this information may be used to understand the progression and prevention of diseases, and to develop new biomaterials for regenerative medicine.

However, in this pursuit, Muhammad was encountering challenges with traditional protein detection methods like the Western blot. As Muhammad puts it, “we were having a problem to produce good quality Western blot images, which consequently affected the accuracy of data processing in our research findings.” And like the signaling pathways he studies, this was a signal that triggered him to respond differently. This pathway led him to Simple Western™, an automated, capillary-based Western assay that has many advantages for studying signaling pathways.

Taking the complexity out of cell signaling

Signaling networks are complex and typically involve many different proteins as well as their phosphorylated isoforms. Gaining an accurate quantitative understanding of signaling networks as a whole is crucial and remains a daunting challenge. The limitations of traditional methods like the Western blot in this regard are significant because they lack throughput, multiplexing efficiency, sensitivity, and quantitation to gain a holistic view of cell signaling.

In stark contrast, Simple Western alleviates these challenges, making it ideally suited for a comprehensive analysis of signaling pathways. For example, Simple Western offers detection in multiple channels, including chemiluminescence and fluorescence channels, and it can even perform two sequential immunoassays in the same capillary with the RePlex™ assay module. These features enable a high level of multiplexing, also called superplexing, to detect many target per sample and lowering the cost per result. Thus, multiplexing with Simple Western can analyze many of the different protein targets in a signaling pathway simultaneously in a single run. Indeed, Muhammad noted how the multiplexing of Simple Western has allowed him to “screen the expression of multiple target proteins at one time”.

Accurate quantification of phosphorylated isoforms

The quantification of phosphorylated isoforms is an important part of cell signaling research. Accurate quantification entails normalizing protein expression to a loading control like total protein content. For this reason, Muhammad uses the Protein Normalization Assay Module for Jess. This assay uses a proprietary molecule to stain the total protein content and automatically normalize target protein expression data in the same capillary. Because it uses a separate channel, it does not interfere with the analysis in the existing IR, NIR and chemiluminescence channels. Furthermore, it eliminates the need for a housekeeping protein as a loading control, which are known to be unreliable loading controls. With this feature, Muhammad explains that “the detection of phospho-protein expression changes during endothelial-to-mesenchymal transition after induced with bioactive compound were obtained.”

Western automation results in quality and reproducibility

Simple Westerns are fully automated. Following a simple sample preparation step, all subsequent steps are precisely executed inside the Simple Western instrument without user intervention, and this results in reproducible and high-quality data. “In terms of results, we are able to get a good quality with high accuracy data,” he explains. And it saves precious time. “Protein analysis using [Simple Western] can be performed faster, eliminating tedious work and saving time.” This frees up time for Muhammad to enjoy hobbies like watching movies. And with all the advanced features of Simple Western, Muhammad says, “I recommended this technology to all my colleagues and collaborators within and outside UKM.”