beta3 Human Adrenoceptor GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

HEK-293 cells stably transfected with a plasmid encoding the human adrenergic b3 receptor are used to prepare membranes in modified Tris-HCl buffer pH 7.4. A 5 μg‡ aliquot of membrane is incubated with 0.5 nM [¹²⁵I] Iodocyanopindolol for 90 minutes at 25℃. Non-specific binding is estimated in the presence of 1 mM alprenolol. Membranes are filtered and washed, the filters are then counted to determine [¹²⁵I] Iodocyanopindolol 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:

[¹²⁵I] Cyanopindolol

Ligand Kd (nM):

1.5

Ligand Concentration:

0.5 nM

Non Specific:

1000 µM Alprenolol

Incubation:

90 min at 25°C

Control Inhibitor:

Alprenolol

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