Cav2.2 (N-type) Rat Calcium Ion Channel Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [¹²⁵I]Conotoxin GVIA to calcium channel N-Type receptors. Frontal lobe brain membranes of male Wistar derived rats weighing 175 ± 25 g are prepared in modified Tris-HCl pH 7.4 buffer using standard techniques. A 40 μg‡ aliquot of membrane is incubated with 10 pM [¹²⁵I]Conotoxin GVIA for 30 minutes at 4℃. Non-specific binding is estimated in the presence of 100 nM w-conotoxin GVIA. Membranes are filtered and washed 3 times and the filters are counted to determine [¹²⁵I]Conotoxin GVIA 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:

[¹²⁵I] w-Conotoxin GVIA

Ligand Kd (nM):

0.051

Ligand Concentration:

0.01 nM

Non Specific:

0.1 µM w-Conotoxin GVIA

Incubation:

30 min at 4°C

Control Inhibitor:

w-Conotoxin GVIA

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