Authors
Hidemasa Goto, Tohru Kiyono, and Masaki Inagaki
Summary
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 its spatially and temporally distribution but also to speculate its 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). To further examine biological roles on the site-specific phosphorylation of a target protein, this protocol includes a method to replace endogenous protein to exogenous phosphorylation-site mutant by both retrovirus-mediated gene transfer and RNA interference (RNAi). Each part of the protocol can be completed in 2-3 months.Further details
The protocol was published on Protocol Exchange in 2007. To see the entire protocol, click on the source link.Advertisement
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