D1 Human Dopamine GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H]SCH-23390 to human dopamine D1 receptors. CHO cells stably transfected with a plasmid encoding the human dopamine D1 receptor are used to prepare membranes in modified Tris-HCl pH 7.4 buffer using standard techniques. A 20 μg‡ aliquot of membrane is incubated with 1.4 nM [³H]SCH-23390 for 120 minutes at 37℃. Non-specific binding is estimated in the presence of 10 µM (+)-butaclamol. Membranes are filtered and washed 3 times and the filters are counted to determine [³H]SCH-23390 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] SCH-23390

Ligand Kd (nM):

1.4

Ligand Concentration:

1.4 nM

Non Specific:

10 µM (+)-Butaclamol

Incubation:

120 min at 37°C

Control Inhibitor:

R(+)-SCH-23390

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