Authors
Hidetaka Kosako
Summary
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 distribution1. 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 phosphorylation2. 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.Further details
The protocol was published on Protocol Exchange on 22 September 2009. To see the entire protocol, click on the source link.Advertisement
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