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Assay Method Information

Assay Name:  Calcium Mobilization Assay
Description:  Briefly, Chinese hamster ovary cells stably expressing the photoprotein aequorin were transiently transfected with WT or respective mutant PKR2-expressing plasmids. Two days after transfection, the cells were charged in Opti-MEM (Invitrogen) containing 8 μM coelenterazine cp (Invitrogen) at 37 °C for 2 h. Cells were detached by brief trypsinization and maintained in assay buffer (Hanks' balanced salt solution plus 10 mM HEPES, pH 7.5, and 0.1% bovine serum albumin) at approximately 5 × 10^5 cells/ml. Luminescence measurements were made using a Sirius Single Tube Luminometer (Berthold Detection Systems GmbH, Pforzheim, Germany). PK2 was serially diluted in assay buffer and mixed with cells. The luminescence was monitored for 35 s (peak 1), then 100 μl of 1% Triton X-100 was injected to lyse the cells, and the luminescence was monitored continuously for another 20 s (peak 2). The area under the curve of the peak 1 was divided by the total area under the curve of peaks 1 and 2, and the maximal responses from WT receptors were normalized to 100.
Affinity data for this assay
 

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Last update November 1, 2007
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