Intein-mediated peptide arrays for epitope mapping and kinase/phosphatase assays.
Synthetic peptides are widely used for production and analysis of antibodies as well as in the study of protein modification enzymes. To circumvent the technical challenges of the existing techniques regarding peptide quantization and normalization, a new method of producing peptide arrays has been developed. This approach utilizes intein-mediated protein ligation that involves linkage of a carrier protein possessing a reactive carboxyl-terminal thioester to a peptide with an amino-terminal cysteine through a native peptide bond. Ligated protein substrates or enzyme-treated samples are arrayed on nitrocellulose membranes with a standard dot-blot apparatus and analyzed by immunoassay. This technique has improved sensitivity and reproducibility, and is suitable for various peptide-based applications. In this report, several experimental procedures including epitope mapping and the study of protein modifications were described.
Xu
Ming-Qun
MQ
New England Biolabs, Beverly, MA, USA.
Ghosh
Inca
I
Kochinyan
Samvel
S
Sun
Luo
L
D000409:Alanine; D000595:Amino Acid Sequence; D045163:Antibodies, Phospho-Specific / Q000494:pharmacology; D000918:Antibody Specificity; D002352:Carrier Proteins / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D018604:Epitope Mapping / Q000379:methods*; D006801:Humans; D015151:Immunoblotting; D047668:Inteins; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry; D010749:Phosphoprotein Phosphatases / Q000378:metabolism*; D010770:Phosphotransferases / Q000378:metabolism*; D040081:Protein Array Analysis / Q000379:methods*; D013379:Substrate Specificity / Q000187:drug effects
D045163:Antibodies, Phospho-Specific; D002352:Carrier Proteins; D010455:Peptides; D010770:Phosphotransferases; D010749:Phosphoprotein Phosphatases; D000409:Alanine