PKA Human AGC Kinase Enzymatic Radiometric LeadHunter Assay [10 μM ATP] - TW
OVERVIEW
DOCUMENTS/REFERENCES
Biological Information
Background Information:
Cyclic-AMP exerts its effects in animal cells mainly by activating the enzyme cyclic AMP-dependent protein kinase A (PKA). This enzyme catalyzes transfer of the terminal phosphate group from ATP to specific serines and threonines of selected proteins. Covalent phosphorylation of the appropriate amino acids of specific cellular proteins in turn results in altered cellullar function as a consequence of activation or inactivation of enzymes or altered membrane permeability to ions or other possible effects. PKA is found in all animal cells, and it is known that hormones or transmitters may be mediated by cAMP-stimulated phosphorylation of specific sites on neuronal or non-neuronal cells to exert their physiological function.
Family:
Kinase
Sub Family:
AGC
Class:
Protein Ser/Thr
Protein Name:
PKACalpha
Uniprot Number:
P17612
Gene Name:
PRKACA
Gene ID:
5566
Gene Aliases:
PKACa
Accession Number:
NM_002730
Organism:
Human
Construct Details:
Fragment, Recombinant
Assay Information
Assay Type:
Biochemical
Assay Sub Type:
Enzymatic
Functional Mode:
Antagonist
Detection Method:
Radiometry
Measured Response:
Scintillation
Testing Information
Procedure Summary:
Human protein kinase PKA expressed in E. coli. is used. Test compound and/or vehicle is preincubated with 60.7 ng/ml enzyme in modified MOPS buffer pH 7.2 at 37°C for 15 minutes. The reaction is initiated by addition of 0.5 µM PKA substrate peptide, 10 µM ATP and 0.05 µCi [γ-³²P]ATP for another 30 minute incubation period and terminated by further addition of 3 % H₃PO₄. An aliquot is removed to a 96 well multiscreen, washed with H₃PO₄ solution, dried and counted to determine the amount of ³²2P- PKA substrate peptide formed. Compounds are screened at 10 µM.
Substrate:
0.5 µM PKA substrate peptide
ATP Concentration:
10 uM
Incubation:
30 min at 37°C
Control Inhibitor:
1-(5-Isoquinolinylsulfonyl)-2-Methylpiperazine
Criteria For Significance:
≥ 50% of max stimulation or inhibition
Test Concentration / Dose:
Suggested concentration for initial testing is 10 uM
Test Sample Requirements:
Minimum for 1) Screen: 25 μl of 10 mM stock -OR- 1 mg (pre-weighed). 2) Dose Response: 60 μl of 10 mM stock -OR- 1 mg (pre-weighed).
Minimum Order Quantity:
2
Turnaround Time
Standard:
15 Business Days
Reference Compound Data
Additional Information
Brand:
LeadHunter
Testing Location:
Taiwan - Taipei
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