EP1 Human Prostanoid GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant prostanoid EP1 receptors expressed in HEK293 cells are used in modified MES buffer pH 6.0. A 14 μg‡ aliquot is incubated with 1 nM [³H]PGE2 for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 10 μM PGE2. Membranes are filtered and washed, the filters are then counted to determine [³H]PGE2 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] Prostaglandin E2 (PGE2)

Ligand Kd (nM):

5.2

Ligand Concentration:

1 nM

Non Specific:

10 µM Prostaglandin E2 (PGE₂)

Incubation:

60 min at 25°C

Control Inhibitor:

Prostaglandin E2

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