ERalpha Human Estrogen NHR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant Estrogen a (ERa) receptors expressed in Sf9 insect cells are used in modified Tris-HCl buffer pH 7.4. A 9.6 ng‡ aliquot is incubated with 0.5 nM [³H] Estradiol for 2 hours at 25°C. Non-specific binding is estimated in the presence of 1 µM diethylstilbestrol. Receptor proteins are filtered and washed, the filters are then counted to determine [³H] Estradiol specifically bound. Compounds are screened at 10 µM.
Note: ‡Membrane protein may change from lot to lot, the concentration used will be adjusted if necessary.

Ligand:

[³H] Estradiol

Ligand Kd (nM):

0.2

Ligand Concentration:

0.5 nM

Non Specific:

1 µM Diethylstilbestrol

Incubation:

120 min at 25°C

Control Inhibitor:

Diethylstilbestrol

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