Phosphoamino acid analysis

Technique

A protein is first phosphorylated using 32P-labeled ATP, usually via an in vitro kinase assay. Most of the amino acids in the protein are then hydrolyzed, usually by the use of a strong acid such as hydrochloric acid. These amino acids are then separated using 2-dimensional thin layer chromatography, along with amino acid standards for the three amino acids that are phosphorylated in eukaryotes: serine, threonine, and tyrosine. These amino acid standards can be visualized on the TLC substrate by exposure to ninhydrin, which colors the amino acids a visible purple when heated at ~100 °C. The radioactive amino acids can be detected via autoradiography, and an overlay of the two images will show which amino acids are phosphorylated.

References

• Rothberg, P.G., Harris, T.J.R., Nomoto, A., and Wimmer, E. (1978) O4-(5'-uridylyl)tyrosine is the bond between the genome-linked protein and the RNA of poliovirus. Proc. Natl. Acad. Sci. USA 75, 4868-4872.
• Eckhart, W., Hutchinson, M.A., and Hunter, T. (1979) An activity phosphorylating tyrosine in polyoma T antigen immunoprecipitates. Cell 18, 925-933.