NET Human Norepinephrine Transporter Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant norepinephrine transporters expressed in dog kidney MDCK cells are used in modified Tris-HCl buffer pH 7.4. A 10 µg‡ aliquot is incubated with 0.2 nM [¹²⁵I]RTI-55 for 3 hours at 4°C. Non-specific binding is estimated in the presence of 10 µM desipramine. Transporters are filtered and washed. The filters are then counted to determine [¹²⁵I]RTI-55 specifically bound. Compounds are screened at 10 µM. ‡ Membrane protein may change from lot to lot; the concentration used will be adjusted if necessary.

Ligand:

[¹²⁵I] RTI-55

Ligand Kd (nM):

24

Ligand Concentration:

0.2 nM

Non Specific:

10 µM Desipramine

Incubation:

180 min at 4°C

Control Inhibitor:

Desipramine

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