A Peptide Microarray for Detecting Protein Kinase Activity in Cell Lysates
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 Zn2+ 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.
- 上一篇
Using Hidden Markov Models to Discover New Protein Transport Machines
Protein import and export pathways are driven by protein tra...
- 下一篇
Reconstitution of Active and Stoichiometric Multisubunit Lysine Acetyltransferase Complexes in Insec
Protein lysine acetyltransferases (KATs) catalyze acetylatio...