View source for Biology:Phosphoamino acid analysis

'''Phosphoamino acid analysis''', or PAA, is an experimental technique used in [[Biology:Molecular biology|molecular biology]] to determine which amino acid or acids are phosphorylated in a protein.
==Technique==
A protein is first phosphorylated using <sup>32</sup>P-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 [[Chemistry:Hydrochloric acid|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: [[Chemistry:Serine|serine]], [[Chemistry:Threonine|threonine]], and [[Chemistry:Tyrosine|tyrosine]]. These amino acid standards can be visualized on the TLC substrate by exposure to [[Chemistry:Ninhydrin|ninhydrin]], which colors the amino acids a visible purple when heated at ~100&nbsp;°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.

[[Category:Protein methods]]

{{Sourceattribution|Phosphoamino acid analysis}}