Detection of ERK activation by a novel monoclonal antibody.

The mitogen-activated protein kinase, ERK is activated by a dual phosphorylation on threonine and tyrosine residues. Using a synthetic diphospho peptide, we have generated a monoclonal antibody directed to the active ERK. The antibody specifically identified the active doubly phosphorylated, but not the inactive mono- or non- phosphorylated forms of ERKs. A direct correlation was observed between ERK activity and the intensity in Western blot of mitogen-activated protein kinases from several species. The antibody was proven suitable for immunofluorescence staining, revealing a transient reactivity with ERKs that were translocated to the nucleus upon stimulation. In conclusion, the antibody can serve as a useful tool in the study of ERK signaling in a wide variety of organisms.