Production of a site- and phosphorylation state-specific antibody.

Hidemasa Goto, Masaki Inagaki

Division of Biochemistry, Aichi Cancer Center Research Institute, Nagoya, Aichi 464-8681, Japan.

Nature protocols 2007

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Protein phosphorylation plays important roles in various aspects of cellular events. Visualization of site-specific phosphorylation in cells is of great importance not only to analyze spatial and temporal distribution but also to investigate biological function. Now, site- and phosphorylation state-specific antibodies are widely utilized as the most powerful tools for these analyses. This protocol details a method to produce the polyclonal version of such an antibody by immunizing a synthetic phosphopeptide corresponding to a protein phosphorylated at targeted site(s). This protocol is also applicable to the production of other types of antibodies, which specifically recognize the site-specific modification, such as acetylation, methylation and proteolysis. The protocol can be completed in 2-3 months.