The peptide microarray-based assay for kinase functionality and inhibition study.
We report a microarray format for the detection of kinase functionality/inhibition based on marking peptide phosphorylation/biotinylation events by the attachment of gold nanoparticles followed by silver deposition for signal enhancement. The detection principle is resonance light scattering (RLS) or surface-enhanced Raman spectroscopy (SERS). Alpha-catalytic subunit of cyclic adenosine 5'-monophosphate (cAMP) dependent protein kinase (PKA) and its well-known substrate, kemptide, are used for the purpose of monitoring phosphorylation and inhibition. As expected, highly selective inhibition of PKA is demonstrated with the four inhibitors: H89, HA1077, mallotoxin and KN62. Furthermore, inhibition assay with inhibitors demonstrate the ability to detect kinase inhibition as well as derive IC50 (half maximal inhibitory concentration) plots.
Wang
Zhenxin
Z
State Key Laboratory of Electroanalytical Chemistry, Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Changchun, China.
D000818:Animals; D057075:Enzyme Assays / Q000379:methods*; D057166:High-Throughput Screening Assays / Q000379:methods; D006801:Humans; D008954:Models, Biological; D019151:Peptide Library; D010455:Peptides / Q000032:analysis; D010766:Phosphorylation; D040081:Protein Array Analysis / Q000379:methods*; D047428:Protein Kinase Inhibitors / Q000032:analysis*; D011494:Protein Kinases / Q000378:metabolism* / Q000502:physiology*
D019151:Peptide Library; D010455:Peptides; D047428:Protein Kinase Inhibitors; D011494:Protein Kinases