H2 Human Histamine GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant histamine H2 receptors expressed in CHO-K1 cells are prepared in K-Na phosphate buffer pH 7.4. A 0.4 µg‡ aliquot is incubated with 0.1 nM [¹²⁵I]Aminopotentidine for 120 minutes at 25°C. Non-specific binding is estimated in the presence of 3 µM Tiotidine. Receptors are filtered and washed, the filters are then counted to determine [¹²⁵I]Aminopotentidine specifically bound. Compounds are screened at 10 µM. ‡Membrane protein may change from lot to lot, the concentration used will be adjusted if necessary.

Ligand:

[¹²⁵I] Aminopotentidine

Ligand Kd (nM):

0.45

Ligand Concentration:

0.1 nM

Non Specific:

3 µM Tiotidine

Incubation:

120 min at 25°C

Control Inhibitor:

Tiotidine

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