A dual array-based approach to assess the abundance and posttranslational modification state of signaling proteins.

A system-wide analysis of cell signaling requires detecting and quantifying many different proteins and their posttranslational modification states in the same cellular sample. Here, we present Protocols for two miniaturized, array-based methods, one of which provides detailed information on a central signaling protein and the other of which provides a broad characterization of the surrounding signaling network. We describe a bead-based array and its use in characterizing the different forms and functions of β-catenin, as well as lysate microarrays (reverse-phase protein arrays) and their use in detecting and quantifying proteins involved in the canonical and noncanonical Wnt signaling pathways. As an application of this dual approach, we characterized the state of β-catenin signaling in cell lysates and linked these molecule-specific data with pathway-wide changes in signaling. The Protocols described here provide detailed instructions for cell culture methods, bead arrays, and lysate microarrays and outline how to use these complementary approaches to obtain insight into a complex network at a systems level.