Using phospho-motif antibodies to determine kinase substrates

Curr Protoc Mol Biol. 2013 Jan;Chapter 18:Unit 18.20.. doi: 10.1002/0471142727.mb1820s101.


Phosphorylation of substrates by protein kinases regulates a myriad of cellular processes, ranging from proliferation and migration to autophagy, senescence, and apoptosis. Kinase substrate selectivity is largely dependent on the amino acid sequence surrounding the phosphorylation site; therefore, substrate-directed, phosphorylation-state-sensitive, motif-specific ("phospho-motif") antibodies represent powerful tools to identify novel kinase substrates and to investigate mechanisms of substrate phosphorylation in many signaling pathways typically associated with human malignancies. Phospho-motif antibodies are engineered to recognize proteins that contain a phosphorylated residue in the context of a specific motif. They are raised against a library of phospho-peptides comprising both the phosphorylated residue and the surrounding residues that determine kinase specificity, with degenerate residues taking up the remaining positions. Currently, several categories of phospho-motif antibody are commercially available, which may be used to specifically detect Ser, Thr, Ser/Thr, or Tyr residues phosphorylated by different protein kinase families. These antibodies are commonly used in immunoprecipitation and/or immunoblotting protocols to determine kinase-induced substrate phosphorylation. This unit describes the use of phospho-motif antibodies to elucidate the kinase(s) responsible for phosphorylating substrate proteins.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Motifs
  • Animals
  • Antibodies / analysis*
  • Electrophoresis, Polyacrylamide Gel / methods
  • HeLa Cells
  • Humans
  • Immunoblotting / methods
  • Immunoprecipitation / methods
  • Phosphopeptides / analysis
  • Phosphopeptides / immunology
  • Phosphopeptides / metabolism
  • Phosphorylation
  • Protein Kinases / metabolism*
  • Substrate Specificity


  • Antibodies
  • Phosphopeptides
  • Protein Kinases