AT1 Human Angiotensin GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant angiotensin AT1 receptors expressed in CHO-K1 cells are used in modified Tris-HCl buffer pH 7.4. A 0.3 μg‡ aliquot is incubated with 0.02 nM [¹²⁵I] (Sar¹, Ile⁸)-Angiotensin II for 180 minutes at 37°C. Non-specific binding is estimated in the presence of 10 µM Angiotensin II. Membranes are filtered and washed, the filters are then counted to determine [¹²⁵I] (Sar¹, Ile⁸)-Angiotensin II 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] (Sar¹, Ile⁸)-Angiotensin II

Ligand Kd (nM):

0.056

Ligand Concentration:

0.02 nM

Non Specific:

10 µM Angiotensin II

Incubation:

180 min at 37°C

Control Inhibitor:

Saralasin

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