alpha2C Human Adrenoceptor GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant adrenergic α2C receptor expressed in CHO-K1 cells are used in modified Tris-HCl buffer pH 7.4. A 2 μg‡ aliquot of membrane is incubated with 0.5 nM [³H]Rauwolscine for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 10 μM WB-4101. Membranes are filtered and washed, the filters are then counted to determine [³H]Rauwolscine 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] Rauwolscine

Ligand Kd (nM):

0.4

Ligand Concentration:

0.5 nM

Non Specific:

10 µM WB-4101

Incubation:

60 min at 25°C

Control Inhibitor:

Yohimbine

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