Accurate Absolute Quantitation of Endogenous AKT1 in Jurkat Cells Using Simple Western

• Published in: Journal of biomolecular techniques 2014-05, Vol.25 (Suppl), p.S28-S28
• Main Authors: Tu, A., Nguyen, U., Kazakova, I., Ramirez, F., Xu, H., Proctor, J., Boge, A., Whaley, P.
• Format: Article
• Language: English
• Subjects: Poster Abstracts
• ISSN: 1524-0215; 1943-4731

Why do researchers use Western blots? It's been the default technology for confirming the presence or absence of a protein, but Western blots are a poor method for measuring the amount of that protein. Accurate quantitation of proteins using traditional Western blotting has been a goal since the technique was developed over thirty years ago. However, because the process requires many steps, each introducing variability, it has not been possible. Portions of the technique have been automated to try and improve consistency, but until now, there has been no major leap in the technology that would propel this method of protein analysis from qualitative to quantitative. Simple Western is the modern evolution of traditional immunoassay techniques. Wes, the latest addition to the Simple Western platform, is an easy to use, fully automated system that removes variability seen with traditional Westerns, so results are more reproducible run to run, between users and over time. The curve-fit feature in the software provided with Wes allows comparison of endogenous proteins in a sample against a standard curve. Researchers are able to not only identify their protein, but also achieve reliable quantitation of their target proteins. To demonstrate how the precision and data reliability of Wes results in more accurate absolute quantitation, we ran spiked GST-tagged AKT1, an oncogene that plays an integral role in triggering the anti-apoptotic response of the PI3K signaling pathway, into a Jurkat cell lysate to act as a standard curve. The spiked lysate was then run on both Wes and a traditional Western blot. When the two methods are compared, Wes not only reduces the hands on time and the time to results, but the reproducibility of the data produces a high degree of confidence in the accuracy of the reported concentration of endogenous AKT1.