title: A peptide microarray for detecting protein kinase activity in cell lysates.
abstract: Protein kinases (PKs) are widely recognized as valuable targets for disease diagnosis and drug discovery. For this reason, we have developed a sensitive peptide microarray for detecting intracellular PK activity. Peptides are immobilized on a glutaraldehyde-premodified high-amino terminal glass slide, by spotting 2 nL volumes of substrate peptide solutions with an automated microarray spotter. After the peptides are phosphorylated by cell lysates, phosphorylation is specifically recognized by a fluorescence-labeled antiphosphotyrosine antibody for tyrosine kinases, or Phos-tag biotin (a biotinylated phosphate-specific ligand based on Zn(2+) complex), which is subsequently bound with fluorescence-labeled streptavidin, for serine/threonine kinases. The fluorescence signal is then detected by an automatic microarray scanner. The peptide microarray system involves simple peptide immobilization, requires low sample volumes and provides a high density array. Importantly, it provides high sensitivity for detecting PK activities in cell lysates. Thus, the peptide microarray system is expected to be useful for a high-throughput kinase assay to investigate intracellular kinase activity and has potential applications in disease diagnosis and drug discovery.
authors.affiliation: Graduate School of System Life Sciences, Kyushu University, Fukuoka, Japan.
authors.forename: Xiaoming
authors.initials: X
authors.lastname: Han
authors.forename: Yoshiki
authors.initials: Y
authors.lastname: Katayama
mesh_terms: D000818:Animals; D002457:Cell Extracts / Q000737:chemistry*; D045744:Cell Line, Tumor; D002471:Cell Transformation, Neoplastic; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays; D006801:Humans; D042541:Intracellular Space / Q000201:enzymology; D008297:Male; D051379:Mice; D055616:Microtechnology; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism*; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D002457:Cell Extracts; D010446:Peptide Fragments; D011494:Protein Kinases