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Ultrasensitive western blot and AlphaScreen® SureFire® assays – complementary tools for studying cellular kinase signaling pathways

Western blotting is widely used for studies of cellular kinases in signaling pathways because it provides high specificity by combining separation with immunodetection. However these assays suffer from limited sensitivity and throughput. Here we demonstrate that an ultra‐sensitive western blot substrate (Western Lightning Ultra) increased detection sensitivity 5‐10 fold, enabling much more robust detection of phosphorylated targets in cellular extracts while significantly reducing consumption of primary antibodies. We further compared the detection of multiple phosphorylated targets using AlphaScreen SureFire assays for cellular kinase activity. Our data indicated that the AlphaScreen SureFire assays for several targets in the IP3 signaling pathway gave results comparable in sensitivity to ultra‐sensitive western blots, but with higher throughput and a much less time‐consuming protocol. We further demonstrated how this assay platform can be used to quantify phosphorylation levels of Akt as a percentage of total Akt protein, and to normalize phosphorylation data using GAPDH protein as a control for variations in cell number.