title: [Kinase-Glo luminescent kinase assay for in vitro determination of PKA activity].
abstract: OBJECTIVE
  To establish a simple, convenient and harmless non-radioactive method of determining protein kinase A (PKA) activity in vitro.
  
  
  METHODS
  Human PKAα cDNA was amplified from total RNA of HEK293 cells using RT-PCR method and then cloned into pGEX-6p-1 vector. In vitro purified GST-PKAα protein was identified by Western blot analysis. Finally, a non-radioactive method, Kinase-Glo luminescent kinase assay, was employed to determine the kinase activity of purified GST-PKAα.
  
  
  RESULTS
  After the optimization of the induction conditions, we purified GST-PKAα protein successfully. We then determined GST-PKAα activity using Kinase-Glo luminescent kinase assay. We also further confirmed the kinase activity of GST-PKAα using H-89, a specific PKA inhibitor, and determined its IC(50); value (35.2±3.97) nmol/L  which is consistent with reported value.
  
  
  CONCLUSIONS
  The non-radioactive Kinase-Glo luminescent kinase assay is a simple, convenient and harmless method of determining the kinase activity of PKA. This method is effective for pre-clinical high-throughput screening of PKA inhibitor, discovering novel target proteins of PKA and investigating PKA phosphorylation sites in target  proteins.
authors: National Institutes for Food and Drug Control, Beijing 100050, China. zhyuan@nicpbp.org.cn
authors: Yuan
authors: Y
authors: Zhang
authors: Yan-lin
authors: YL
authors: Wu
authors: Qian
authors: Q
authors: Liu
authors: Chen
authors: C
authors: Chen
authors: Yuan-yuan
authors: YY
authors: Qin
authors: Gui-yan
authors: GY
authors: Wu
authors: Hua
authors: H
authors: Gao
mesh_terms: D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006801:Humans; D020128:Inhibitory Concentration 50; D007546:Isoquinolines / Q000494:pharmacology; D008163:Luminescent Measurements / Q000379:methods*; D010766:Phosphorylation / Q000187:drug effects; D010957:Plasmids / Q000235:genetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D013449:Sulfonamides / Q000494:pharmacology
chemical_list: D007546:Isoquinolines; D047428:Protein Kinase Inhibitors; D011993:Recombinant Fusion Proteins; D013449:Sulfonamides; D017868:Cyclic AMP-Dependent Protein Kinases; C063509:N-(2-(4-bromocinnamylamino)ethyl)-5-isoquinolinesulfonamide