D3 Human Dopamine GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H]Spiperone to human dopamine D3 receptors. CHO cells stably transfected with a plasmid encoding the human dopamine D3 receptor are used to prepare membranes in modified Tris-HCl pH 7.4 buffer using standard techniques. A 10 μg‡ aliquot of membrane is incubated with 0.7 nM [³H]Spiperone for 120 minutes at 37℃. Non-specific binding is estimated in the presence of 25 µM S(-)-sulpiride. Membranes are filtered and washed 3 times and the filters are 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):

0.36

Ligand Concentration:

0.7 nM

Non Specific:

25 µM S(-)-Sulpiride

Incubation:

120 min at 37°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