Methods in Enzymology Vol.201 Protein Phosphorylation, Part B: Analysis of Protein Phosphorylation

Discussion in 'Methods in Enzymology Book Series' started by aaronmax, Jul 26, 2016.

  1. aaronmax

    aaronmax New Member

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    This chapter discusses the separation of phosphotyrosine, phosphoserine, and phosphothreonine by high-performance liquid chromatography (HPLC). A fast, sensitive, HPLC method for analyzing the products of protein kinase-catalyzed phosphorylations of the three hydroxyamino acids, tyrosine, serine, and threonine, has been developed. This method incorporates the use of precolumn derivatization of amino acids with the fluorescent reagent, 9-fluorenylmethyl chloroformate (FMOC), and their subsequent separation on an anion-exchange column under conditions of isocratic elution. The separation and quantitative analysis of FMOC-derivatized phosphorylated hydroxyamino acids by HPLC allow rapid, sensitive detection without the need for 32P-labeling. This is an important feature when working with a sample from an in vivo source where radioisotopic labeling may not be possible or feasible. Some other inherent advantages of this approach for phosphoamino acid analysis are the ability to sample total phosphoamino acid content instead of just labeled forms and the ability to distinguish new phosphorylations and phosphate turnover at preexisting sites.
    • Series: Methods in Enzymology (Book 201)
    • Hardcover: 547 pages
    • Publisher: Academic Press; 1 edition (August 20, 1991)
    • Language: English
    • ISBN-10: 0121821021
    • ISBN-13: 978-0121821029
    • Product Dimensions: 6 x 1.2 x 9 inches
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