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Assay Method Information

Assay Name:  Inhibition Assay
Description:  PDE3 inhibition assay was performed a BIOMOL GREEN Quantizyme Assay System (catalogue No. BML-AK800-0001). The Platelets isolated from human blood were used as a source of PDE3 enzyme. 10 mL blood collected in a vacutainer tube (containing K3 EDTA) and centrifuged at 190xg for 15 min at room temperature. Top layer (platelet rich plasma) collected, centrifuged at 2500 g for 5 min at 22° C. (Room temp.) The pellet was washed with 2 ml of 50 mM tris buffer (pH-7.4) containing 1 mM MgCl2 and centrifuged at 2500 g for 5 min. Then 200 μl of assay buffer (from PDE kit, Enzo Life Sciences) was added to the pellet and sonicated at 30 s per ml. Pellet was freeze thawed for three times (-80° C.) in order to rupture the platelet membrane and release the PDE enzyme. Then the cell homogenate was centrifuged at 2500 rpm for 5 min. Supernatant was collected and used as source for PDE3 enzyme. In 96 well plate (Prod. No. BML-KI101), we added supernatant having PDE3 enzyme, PDE3 assay buffer, cAMP substrate, 5' nucleotidase and test or standard compound and incubated for 1 hour at 37° C. The reaction was arrested by the addition 100 μl BIOMOL GREEN reagent incubated in room temp for 20 min.
Affinity data for this assay
 

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Last update November 1, 2007
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