CYP19A1 Human CYP450 Enzymatic LeadHunter Assay - TW

Procedure Summary:

Human recombinant CYP450 19 plus P450 reductase, expressed in baculovirus infected insect BTI-TN-5B1-4 cells is used. Test compound and/or vehicle is preincubated with 4 nM enzyme‡ in phosphate buffer pH 7.4 for 15 minutes at 37°C. The reaction is initiated by addition of 0.8 μM dibenzylfluorescein and cofactor solution (1.3 mM NADP, 3.5 mM glucose-6-phosphate, 3.3 mM MgCl2 and 0.4 U/ml G6P dehydrogenase) for another 45 minutes and terminated by further addition of 1N NaOH. Determination of the amount of dibenzylfluorescein remaining is read spectrofluorimetrically. Compounds are screened at 10 μM.

Substrate:

0.8 µM Dibenzylfluorescein

Incubation:

45 min at 37°C

Control Inhibitor:

Testosterone

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