| Assay Method Information | |
| | Aequorin-Based Luminescent Assay |
| Description: | An aequorin-based luminescent assay for calcium mobilization was used to measure mobilization of intracellular Ca2+ (Bullock et al., Mol Pharmacol 65, 582-588, 2004). Chinese hamster ovary (CHO) cells stably expressing photoprotein aequorin and recombinant PKR1 or PKR2 were tested by this method. Briefly, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM of coelenterazine cp at 37° C. for 2 hours. Cells were detached by brief trypsinization and maintained in Hank's Balanced Salt Solution (HBSS) plus 10 mM HEPES (pH7.5) and 0.1% BSA at about 5×105 cells/ml. Luminescence measurements were made using a Berthold luminometer.All compounds were diluted in HBSS plus 10 mM HEPES (pH7.5) and 0.1% BSA. To test the agonist activity, 100 μl of cells were injected into the tubes with 20 μl of compounds. For antagonist assays, 80 μl cells were incubated in the tubes with 20 μl different concentrations of antagonists at room temperature for 20 minutes, and then 100 μl of recombinant PK2 were injected. The IC50 obtained from the assays were then converted to Ki values using the formula: IC50/(1+[PK2]/EC50PK2). |
| Affinity data for this assay | |
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