D4.4 Human Dopamine GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

CHO-K1 cells stably transfected with a plasmid encoding the human dopamine D4.4 receptor are used to prepare membranes in modified Tris-HCl buffer pH 7.4. A 100 μg‡ aliquot of membrane is incubated with 1.2 nM [³H]Spiperone for 120 minutes at 25℃. Non-specific binding is estimated in the presence of 10 µM haloperidol. Membranes are filtered and washed, the filters are then counted to determine [³H]Spiperone 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] Spiperone

Ligand Kd (nM):

0.46

Ligand Concentration:

1.2 nM

Non Specific:

10 µM Haloperidol

Incubation:

120 min at 25°C

Control Inhibitor:

Spiperone

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