UNIT 18.14 Analysis of Serine-Threonine Kinase Specificity Using Arrayed Positional Scanning Peptide Libraries
Published Online: 1 JUL 2010
Copyright © 2010 by John Wiley & Sons, Inc.
Lab Protocol Title
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.
- Published Online: 1 JUL 2010
- Published Print: JUL 2010
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.
- phosphorylation motif;
- kinase specificity;
- peptide array