Unit

UNIT 18.14 Analysis of Serine-Threonine Kinase Specificity Using Arrayed Positional Scanning Peptide Libraries

  1. Catherine Chen,
  2. Benjamin E. Turk

Published Online: 1 JUL 2010

DOI: 10.1002/0471142727.mb1814s91

Current Protocols in Molecular Biology

Current Protocols in Molecular Biology

How to Cite

Chen, C. and Turk, B. E. 2010. Analysis of Serine-Threonine Kinase Specificity Using Arrayed Positional Scanning Peptide Libraries. Current Protocols in Molecular Biology. 91:18.14:18.14.1–18.14.15.

Author Information

  1. Yale University, New Haven, Connecticut

Publication History

  1. Published Online: 1 JUL 2010
  2. Published Print: JUL 2010

Abstract

Protein kinases vary substantially in their consensus phosphorylation motifs, the residues that are either preferred or deselected by the kinase at specific positions surrounding the phosphorylation site. The protocol described here is used to rapidly determine phosphorylation motifs for serine-threonine kinases. The procedure involves screening an arrayed combinatorial peptide library consisting of 198 biotinylated substrates. Peptides are phosphorylated by the kinase of interest in the presence of radiolabeled ATP and then captured on streptavidin membrane. The membrane is subsequently washed, dried, and exposed to a phosphor screen to visualize and quantify incorporation of radiolabel into the peptides. The phosphorylation motif is thereby derived from the relative extent of phosphorylation of each peptide in the array. Curr. Protoc. Mol. Biol. 91:18.14.1-18.14.15. © 2010 by John Wiley & Sons, Inc.

Keywords:

  • phosphorylation motif;
  • kinase specificity;
  • peptide array