[Kinase-Glo luminescent kinase assay for in vitro determination of PKA activity].
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi
; 28(7): 684-7, 2012 Jul.
Article
en Zh
| MEDLINE
| ID: mdl-22768854
ABSTRACT
AIM:
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.CONCLUSION:
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.
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Colección:
01-internacional
Banco de datos:
MEDLINE
Asunto principal:
Proteínas Quinasas Dependientes de AMP Cíclico
/
Mediciones Luminiscentes
Límite:
Humans
Idioma:
Zh
Revista:
Xi Bao Yu Fen Zi Mian Yi Xue Za Zhi
Asunto de la revista:
ALERGIA E IMUNOLOGIA
Año:
2012
Tipo del documento:
Article
País de afiliación:
China