Cav1.2 (L-type) Rat Calcium Ion Channel Binding (Verapamil Site) LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H](-)-Desmethoxyverapamil (D-888) to calcium channel Type L, phenylalkylamine receptors. Cerebral Cortex membranes of male Wistar derived rats weighing 175 ± 25 g are prepared in HEPES buffer pH 7.4 buffer using standard techniques. A 10 mg‡ aliquot of membrane is incubated with 0.4 nM [³H](-)-Desmethoxyverapamil for 60 minutes at 25℃. Non-specific binding is estimated in the presence of 10 µM D-600. Membranes are filtered and washed 3 times and the filters are counted to determine [³H](-)-Desmethoxyverapamil 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] (-)-Desmethoxyverapamil (D-888)

Ligand Kd (nM):

14

Ligand Concentration:

0.4 nM

Non Specific:

10 µM Methoxyverapamil (D-600)

Incubation:

60 min at 25°C

Control Inhibitor:

Methoxyverapamil (D-600)

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