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Assay Method Information

Assay Name:  PDE3 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 2500g 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 ul 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 ul BIOMOL GREEN reagent incubated in room temp for 20 min. The green color developed was measured at 620 nm.
Affinity data for this assay
 

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Last update November 1, 2007
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