kappa (KOP) Human Opioid GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human opiate κ receptors expressed in HEK-293 cells are used in modified Tris-HCl buffer pH 7.4. A 30 μg‡ aliquot is incubated with 0.6 nM [³H]Diprenorphine for 60 minutes at 25°C. Nonspecific binding is estimated in the presence of 10 μM naloxone. Membranes are filtered and washed, the filters are then counted to determine [³H]Diprenorphine 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] Diprenorphine

Ligand Kd (nM):

0.4

Ligand Concentration:

0.6 nM

Non Specific:

10 µM Naloxone

Incubation:

60 min at 25°C

Control Inhibitor:

U-69593

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