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Simple Western Analysis of NF-kappaB Signaling Cascade Proteins

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Aberrant expression and signaling of multiple proteins in the NF-κB pathway are commonly associated with inflammatory and stress-induced diseases, including many cancers. Understanding how NF-κB 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 newest member of the Simple Western platform from ProteinSimple. Sally is easy to set-up and runs hands-free up to 96 data points in a single experiment, thus addressing the need for higher throughput. Sally generates 8 individual measures from 5 µL of sample which allows for characterization of whole signaling pathways from one small sample size. Data generated on Sally from examination of targets in the NF-κB (p100/p50) pathway demonstrate high reproducibility and low intrassay variability. Response to TNF-α treatment in whole cell and nuclear extracts of IκB and NF-κB subunits (c-Rel, p65, and p50/p105) demonstrates, as expected, statistically significant changes in signal and localization. Results and workflow comparisons indicate a distinct advantage of the Simple Western when compared to traditional Western methods.

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