||Solid-Phase Assay for the Phosphorylation of Proteins Blotted on Nitrocellulose Membrane Filters.
Valtorta, F. ;
Scheibler, W. ;
Jahn, R. ;
Ceccarelli, B. ;
Greengard, P. ;
||Rockefeller Univ., New York. ;Milan Univ. (Italy).;Health Effects Research Lab., Research Triangle Park, NC.
Protein kinases ;
Peptide mapping ;
Laboratory animals ;
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A new procedure for the phosphorylation and assay of phosphoproteins is described. Proteins are solubilized from tissue samples, separated by polyacrylamide gel electrophoresis, transferred onto nitrocellulose membrane filters and the blotted polypeptides are phosphorylated with the catalytic subunit of cyclic AMP (3', 5'-adenosine monophosphate)-dependent protein kinase. The method was developed for the assay of dephosphosynapsin I, but it has also proven suitable for the phosphorylation of other proteins. The patterns of phosphorylation of tissue samples phosphorylated using the new method are similar to those obtained using the conventional test tube assay. Once phosphorylated, the adsorbed proteins can be digested with proteases and subjected to phosphopeptide mapping. The phosphorylated blotted proteins can also be analyzed by overlay techniques for the immunological detection of polypeptides. (Copyright (c) by Academic Press, Inc.)