GABAA (Non-Selective) Rat Ion Channel [3H] Ro-15-1788 Binding (Cerebellum) LeadHunter Assay - TW

Procedure Summary:

Cerebella of Wistar derived rats weighing 175 ± 25 g are used to prepare GABAA central benzodiazepine membrane receptor in 50 mM Tris-Citrate buffer pH 7.4. A 1.5 mg‡ aliquot is incubated with 1 nM [³H]Ro-15-1788 for 120 minutes at 4℃. 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]Ro-15-1788 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] Ro-15-1788

Ligand Kd (nM):

2

Ligand Concentration:

1 nM

Non Specific:

10 µM Diazepam

Incubation:

120 min at 4°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