Determining protein kinase substrate specificity by parallel solution-phase assay of large numbers of peptide substrates Academic Article uri icon


MeSH Major

  • Molecular Probe Techniques
  • Protein Kinases


  • We describe here a protocol for determining the activity of protein kinases on a large set of peptide substrates. Biotin-tagged peptides are arrayed in multiwell plates and incubated in solution with the kinase of interest and radiolabeled ATP. Reactions are then spotted simultaneously onto a streptavidin membrane, which is washed, dried, and analyzed by autoradiography or phosphor imaging. Differences in the extent of radiolabel incorporation into the various peptide substrates provide a measure of the sequence specificity of the kinase. This approach is a faster, more sensitive, and more generally applicable method for determining kinase phosphorylation motifs than older peptide library screening approaches based on Edman sequencing. The procedure is readily adaptable to other applications that require parallel processing of many kinase reactions, such as screening for small molecule inhibitors. In the format described here, preparation of stock plates prior to running the reactions will require about 4 days. Afterwards, the protocol takes approximately 6 hours to perform.

publication date

  • June 2006



  • Academic Article



  • eng

Digital Object Identifier (DOI)

  • 10.1038/nprot.2006.57

PubMed ID

  • 17406259

Additional Document Info

start page

  • 375

end page

  • 9


  • 1


  • 1