Maximizing TNFα signaling pathway characterization with Simple Western (Neuroscience 2012)

Aberrant expression and signaling of multiple proteins in the NF-KB pathway are commonly associated with inflammatory and stress-induced diseases, including many cancers. Understanding how NF-KB 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 for the first time results generated on Sally, the new Simple Western 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 low inter- and intrassay variability. This suggests the potential to characterize whole signaling pathways with as little as 5 μL of sample. Targets from the NF-κB pathway, including IκB, NF-κB subunits c-Rel, p65, and p50/p105 from both whole cell and nuclear lysates, were screened on the Simple Western platform in response to TNF-α treatment. Statistically significant changes in signal and localization were clearly observed for each of the key targets. Results and workflow comparisons indicate a distinct advantage of the Simple Western when compared to traditional Western methods.