Phos-tag Western blotting for detecting stoichiometric protein phosphorylation in cells

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.