A solid-phase assay for the phosphorylation of proteins blotted on nitrocellulose membrane filters

Flavia Valtorta, Werner Schiebler, Reinhard Jahn, Bruno Ceccarelli, Paul Greengard

Research output: Contribution to journalArticlepeer-review

Abstract

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 (adenosine 3′:5′-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.

Original languageEnglish
Pages (from-to)130-137
Number of pages8
JournalAnalytical Biochemistry
Volume158
Issue number1
DOIs
Publication statusPublished - 1986

Keywords

  • neurochemistry
  • nitrocellulose
  • protein blotting
  • protein kinases
  • protein phosphorylation
  • synapsin I

ASJC Scopus subject areas

  • Biochemistry
  • Biophysics
  • Molecular Biology

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