beta (Non-Selective) Rat Adrenoceptor GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Brains (except cerebella) of male Wistar derived rats weighing 175 ± 25 g are used to prepare adrenergic b receptors in modified Tris-HCl buffer pH 7.4. A 7.5 mg‡ aliquot is incubated with 0.25 nM [³H]Dihydroalprenolol for 20 minutes at 25℃. Non-specific binding is estimated in the presence of 1 µM S(-)-Propranolol. Membranes are filtered and washed, the filters are then counted to determine [³H]Dihydroalprenolol 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] Dihydroalprenolol

Ligand Kd (nM):

0.5

Ligand Concentration:

0.25 nM

Non Specific:

1 µM S(-)-Propranolol

Incubation:

20 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