nAChR (alpha4/beta2) Human Ion Channel Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant nicotinic acetylcholine α4β2 receptors expressed in SH-SY5Y cells are used in modified Tris-HCl buffer pH 7.4. A 7 µg‡ aliquot incubated with 0.6 nM [³H]Cytisine for 120 minutes at 4°C. Non-specific binding is estimated in the presence of 10 µM nicotine. Membranes are filtered and washed. The filters are then counted to determine [³H]Cytisine specifically bound. Compounds are screened at 10 µM. ‡Membrane may change from lot to lot; the concentration used will be adjusted if necessary.

Ligand:

[³H] Cytisine

Ligand Kd (nM):

0.3

Ligand Concentration:

0.6 nM

Non Specific:

10 µM (-)-Nicotine

Incubation:

120 min at 4°C

Control Inhibitor:

Nicotine

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