AI Article Synopsis
- The study aimed to develop a non-radioactive method for measuring protein kinase A (PKA) activity in vitro, making the process simpler and safer.
- Researchers used RT-PCR to amplify human PKAα cDNA from HEK293 cells, then purified the GST-PKAα protein and verified its presence through Western blot analysis.
- The Kinase-Glo luminescent assay was successfully used to assess the kinase activity of GST-PKAα, confirming its effectiveness for potential pre-clinical screening and discovery related to PKA inhibitors and phosphorylation sites.
Article 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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