PDE5A Human Phosphodiesterase Enzymatic LeadHunter Assay - TW

Procedure Summary:

Human recombinant PDE5A expressed in insect Sf9 cells are used. Test compound and/or vehicle is preincubated with 20 ng/ml enzymeó in Tris-HCl buffer pH 7.2 for 15 minutes at 25⁰C. The reaction is initiated by addition of 100 nM fluorescein lableled cGMP for another 30 minutes incubation period and terminated by addition of IMAP binding solution. IMAP complexes with phosphate groups on nucleotide monophosphate generated from cyclic nucleotides through PDE activity. Determination of the amount of complex formed is read spectrofluorimetrically at 470 nm/525 nm. Compounds are screened at 10 µM.

Substrate:

0.1 µM FAM-cGMP

Incubation:

30 min at 25°C

Control Inhibitor:

Zaprinast

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