CCK2 (CCKB) Human Cholecystokinin GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Rat hematopoietic Chem-1 cells stably transfected with a plasmid encoding the human recombinant CCK2 cholecystokinin receptor are used to prepare membranes in modified HEPES buffer pH 7.4. A 0.6 μg‡ aliquot of membrane is incubated with 0.05 nM [¹²⁵I]CCK-8 for 180 minutes at 25°C. Non-specific binding is estimated in the presence of 1 μM Sincalide. Membranes are filtered and washed, the filters are then counted to determine [¹²⁵I]CCK-8 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] CCK-8

Ligand Kd (nM):

0.069

Ligand Concentration:

0.05 nM

Non Specific:

1 µM Sincalide

Incubation:

180 min at 25°C

Control Inhibitor:

Sincalide

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