Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels

Robert L. Geahlen, Michael Anostario, Philip S. Low, Marietta L. Harrison

Research output: Contribution to journalArticle

74 Scopus citations

Abstract

A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [γ-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [γ-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 μg can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.

Original languageEnglish (US)
Pages (from-to)151-158
Number of pages8
JournalAnalytical Biochemistry
Volume153
Issue number1
DOIs
StatePublished - Feb 15 1986

Keywords

  • autoradiography
  • gel electrophoresis
  • protein kinases
  • protein phosphorylation
  • proteins

ASJC Scopus subject areas

  • Molecular Biology
  • Biophysics
  • Biochemistry

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