Using Phospho‐Motif Antibodies to Determine Kinase Substrates

Colin D. White1, Alex Toker1

1 Beth Israel Deaconess Medical Center and Harvard Medical School, Boston, Massachusetts
Publication Name:  Current Protocols in Molecular Biology
Unit Number:  Unit 18.20
DOI:  10.1002/0471142727.mb1820s101
Online Posting Date:  January, 2013
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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.

Keywords: phospho‐motif antibody; phosphorylation; protein kinase

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Table of Contents

  • Introduction
  • Basic Protocol 1: In Silico Analysis to Identify the Kinase Responsible for Phosphorylating a Known Substrate
  • Basic Protocol 2: Biochemical Validation of Protein Kinase–Induced Substrate Phosphorylation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
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Basic Protocol 1: In Silico Analysis to Identify the Kinase Responsible for Phosphorylating a Known Substrate

  • Ligand(s) for cell stimulation (see protocol 2 introduction)
  • 10‐cm2 dish of 90% to 100% confluent cells
  • Phosphate‐buffered saline (PBS; see recipe), ice‐cold
  • RIPA (radio‐immunoprecipitation assay) lysis buffer (see recipe)
  • Modified Bradford assay kit (Bio‐Rad #500‐0002; also see unit 10.1)
  • SDS sample buffer (see recipe)
  • Antibody raised against the putative protein kinase substrate [Cell Signaling Technology (, Abcam (, or Santa Cruz Biotechnology (]
  • Protein A‐Sepharose slurry (Fisher Scientific 45‐000‐116)
  • Immunoprecipitation (IP) wash buffer (see recipe)
  • Phospho‐motif antibody (see Table 18.20.1)
  • 5% nonfat dry milk/TBS‐T (see recipe)
  • 5% BSA/TBS‐T: 5% bovine serum albumin (Sigma‐Aldrich #A7030) in TBS‐T (see recipe), freshly prepared
  • Protein A‐HRP (horseradish peroxidase) (Millipore #18‐160). Immediately prior to use, dilute 1:5,000 in 5% nonfat dry milk/TBS‐T (see recipe)
  • Aspirator
  • Cell scrapers
  • Polyvinylidene fluoride (PVDF) membranes (optional)
  • Additional reagents and equipment for Bradford assay (unit 10.1), SDS‐PAGE (unit 10.2), and immunoblotting (unit 10.8)
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Literature Cited

Literature Cited
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