To investigate the physiological roles of protein phosphorylation, it is important to analyze sites and stoichiometry of phosphorylation in cells. The generation of phosphorylation site-specific antibodies is useful to detect targeted phosphorylation sites and visualize their intracellular distribution (1). However, it is difficult to determine the stoichiometry of phosphorylation by using these antibodies. Phosphate-affinity polyacrylamide gel electrophoresis is useful to detect stoichiometric protein phosphorylation (2). The phosphate-affinity site is a polyacrylamide-bound dinuclear Mn2+ complex (Mn2+-Phos-tag) that can enhance mobility shifts of phosphorylated forms of many proteins. Phosphorylation levels of cellular proteins of interest can be assessed by subsequent Western blotting.