alpha2B Human Adrenoceptor GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H]Rauwolscine to human adrenergic a2B receptor CHO-K1 cells stably transfected with a plasmid encoding the human adrenergic a2B receptor are used in modified Tris-HCl pH 7.4 buffer using standard techniques. A 10 μg‡ aliquot of membrane is incubated with 2.5 nM [³H]Rauwolscine for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 10 μM prazosin. Membranes are filtered and washed 3 times and filters are 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):

2.1

Ligand Concentration:

2.5 nM

Non Specific:

10 µM Prazosin

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