GABAA (Non-Selective) Rat Ion Channel [3H] Flunitrazepam Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Whole brains (except cerebellum) of male Wistar-derived rats weighing 175 ± 25 g are used to prepare GABAA central benzodiazepine receptor in Na-K phosphate buffer pH 7.4. A 2 mg‡ aliquot is incubated with 1 nM [³H]Flunitrazepam for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 10 µM diazepam. Membranes are filtered and washed. The filters are then counted to determine [³H]Flunitrazepam 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:

[³H] Flunitrazepam

Ligand Kd (nM):

4.4

Ligand Concentration:

1 nM

Non Specific:

10 µM Diazepam

Incubation:

60 min at 25°C

Control Inhibitor:

HYDROGEN DIAZEPAM CHLORIDE

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