AR Human Androgen NHR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human androgen receptors obtained from human LNCaP cells are used in modified HEPES buffer pH 7.4. A 70 µg‡ aliquot is incubated with 0.5 nM [³H]Methyltrienolone for 20 hours at 4°C. Non-specific binding is estimated in the presence of 1 µM Methyltrienolone. Receptors are filtered and washed. The filters are then counted to determine [³H]Methyltrienolone specifically bound. Compounds are screened at 10 µM. ‡The concentration used will be adjusted if necessary.

Ligand:

[³H] Methyltrienolone

Ligand Kd (nM):

0.71

Ligand Concentration:

0.5 nM

Non Specific:

1 µM Testosterone

Incubation:

1200 min at 4°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