Sandwich assay for phosphorylation of protein multiplexes by using antibodies and Phos-tag.

We describe two procedures for performing sandwich assays of phosphorylation of protein multiplexes by using several antibodies and a biotinylated Phos-tag with a dodeca(ethylene glycol) spacer. The first procedure is based on an antibody microarray technique with an enhanced chemiluminescence system, and it permits the simultaneous and highly sensitive detection of multiple phosphoproteins in a cell lysate. The second is based on the Bio-Plex suspension array technique with a flow-based microplate fluorescence reader system. By using this procedure, we demonstrated the quantitative detection of the entire level of phosphorylation in a target protein involved in intracellular signaling.