Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein. (June 2004)

• Record Type: Journal Article
• Authors: Xu, Jie; Sun, Luo; Ghosh, Inca; Xu, Ming-Qun
• Is Part Of: Biotechniques. Volume 36:Number 6(2004)
• Page Start: 976
• Page End: 981
• Publication Date: 2004-06
• Subjects: Biology, Experimental -- Periodicals; Molecular biology -- Periodicals; Medical technology -- Periodicals; Biology, Experimental; Medical technology; Molecular biology; Clinical Laboratory Techniques -- Periodicals; Research -- Periodicals; Medical Laboratory Science -- Periodicals; Periodicals; Electronic journals; 570
• DOI: 10.2144/04366ST02
• Languages: English
• ISSNs: 0736-6205

Abstract

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.