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

Procedure Summary:

This assay measures binding of [³H]Diltiazem to calcium channel L-Type benzothiazepine receptors. Brains (except cerebella) of male Wistar derived rats weighing 175 ± 25 g are prepared in modified Tris-HCl pH 7.4 buffer using standard techniques. A 0.5 mg‡ aliquot of membrane protein is incubated with 2 nM [³H]Diltiazem for 180 minutes at 4℃. Non-specific binding is estimated in the presence of 10 µM Diltiazem. Membranes are filtered and washed 3 times and the filters are counted to determine [³H]Diltiazem 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] Diltiazem

Ligand Kd (nM):

16

Ligand Concentration:

2 nM

Non Specific:

10 µM Diltiazem

Incubation:

180 min at 4°C

Control Inhibitor:

Diltiazem

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