BioTechniques (Jun 2004)

Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein

  • Jie Xu,
  • Luo Sun,
  • Inca Ghosh,
  • Ming-Qun Xu

DOI
https://doi.org/10.2144/04366ST02
Journal volume & issue
Vol. 36, no. 6
pp. 976 – 981

Abstract

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We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.