Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein.
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.
Xu
Jie
J
New England Biolabs, Beverly, MA, USA.
Sun
Luo
L
Ghosh
Inca
I
Xu
Ming-Qun
MQ
D000918:Antibody Specificity; D015153:Blotting, Western / Q000379:methods*; D002352:Carrier Proteins / Q000276:immunology*; D006801:Humans; D010455:Peptides / Q000276:immunology*; D011485:Protein Binding; D013379:Substrate Specificity; D019061:src-Family Kinases / Q000032:analysis* / Q000276:immunology*
D002352:Carrier Proteins; D010455:Peptides; D019061:src-Family Kinases