Simple Western analysis of signaling cascade proteins (CE Pharm 2012)

Proteins in the AKT signaling cascade are associated with tumor cell survival, proliferation, and invasiveness. The activation of AKT is also one of the most frequent alterations observed in human cancer and tumor cells. Understanding how AKT signaling impacts disease progression is important to the development of novel therapeutics. Cell signaling events are routinely assessed using traditional Western blot analysis. The Western blot technique is very labor intensive and generally yields results that are semi-quantitative. The Simple Western platform described here completely automates the manual steps involved in traditional Western blot protocols and can analyze up to 96 samples in a single experiment. Because Simple Western protocols consume only µliter sample volumes, reproducible and quantitative results can be generated from precious or quantity-limited samples. We present results generated on Sally, the higher throughput platform from ProteinSimple. Sally is able to run up to 96 data points in a single experiment, addressing the needs for higher throughput applications in screening signaling pathways. Data generated on Sally demonstrates high reproducibility and intrassay variability of less than 10%. Seven targets from the AKT pathway, were screened with both anti -total and anti-phospho antibodies on the Simple Western platform from a total volume of 5 µL/treatment. In response to Insulin treatment, significant and expected signal changes were clearly observed for each of the key targets. This suggests the potential to characterize whole signaling pathways with as little as 5 µL of sample. Results and workflow comparisons indicate a distinct advantage of the Simple Western when compared to traditional Western methods.