Application Note

Simultaneous Detection of Total and Phosphorylated Protein in Cellular Kinase Assay Using Alpha SureFire Ultra Multiplex Technology


Cellular kinase signal transduction pathways are involved in the regulation of many important cellular processes such as cell survival, differentiation, and apoptosis. Kinase signaling networks are typically characterized by multiple kinases arranged in cascades containing nodes with feedback loops and crosstalk between pathways. Numerous assay technologies have been developed for studying kinase signaling pathways, and for screening compound libraries in search of agents to modify receptors or kinase activities. The quality of these assays can be impacted by data variability due to cell seeding inhomogeneity or from compound toxicity. A common method for controlling for variability is to normalize the assay signal to a cellular protein whose level does not change as a function of the treatment.

Alpha SureFire® Ultra Multiplex Phospho and Total assays utilize two types of Alpha Acceptor beads to simultaneously measure two signals from each assay well to easily normalize the assay. Here, we demonstrate the benefit and utility of normalizing assay signal of phosphorylated protein levels to total protein levels in two different cellular models: ERK 1/2 phosphorylation in human melanoma A375 cells and AKT 1/2/3 phosphorylation in mouse myoblast C2C12 cells.