beta1 Human Adrenoceptor GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant adrenergic β1 receptors expressed in HEK 293 cells are used in modified Tris-HCl buffer pH 7.4. A 25 µg‡ aliquot is incubated with 0.03 nM [¹²⁵I]Cyanopindolol for 120 minutes at 25℃. Non-specific binding is estimated in the presence of 100 µM S(-)-Propranolol. Receptors are filtered and washed, the filters are then counted to determine [¹²⁵I]Cyanopindolol specifically bound. Compounds are screened at 10 µM.

Ligand:

[¹²⁵I] Cyanopindolol

Ligand Kd (nM):

0.041

Ligand Concentration:

0.03 nM

Non Specific:

100 µM S(-)-Propranolol

Incubation:

120 min at 25°C

Control Inhibitor:

S(-)-Propranolol

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