A peptide microarray for detecting protein kinase activity in cell lysates.

Xiaoming Han, Yoshiki Katayama

Research output: Contribution to journalArticlepeer-review

11 Citations (Scopus)

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.

Original languageEnglish
Pages (from-to)183-194
Number of pages12
JournalMethods in molecular biology (Clifton, N.J.)
Volume669
DOIs
Publication statusPublished - 2010

All Science Journal Classification (ASJC) codes

  • Molecular Biology
  • Genetics

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