UNIT 18.20 Using Phospho-Motif Antibodies to Determine Kinase Substrates

  1. Colin D. White,
  2. Alex Toker

Published Online: 1 JAN 2013

DOI: 10.1002/0471142727.mb1820s101

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

White, C. D. and Toker, A. 2013. Using Phospho-Motif Antibodies to Determine Kinase Substrates. Current Protocols in Molecular Biology. 101:18.20:18.20.1–18.20.11.

Author Information

  1. Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts

Publication History

  1. Published Online: 1 JAN 2013
  2. Published Print: JAN 2013


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. Curr. Protoc. Mol. Biol. 101:18.20.1–18.20.11. © 2013 by John Wiley & Sons, Inc.


  • phospho-motif antibody;
  • phosphorylation;
  • protein kinase