alpha1D Human Adrenoceptor GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H]Prazosin to adrenergic a1D receptors. Membranes (obtained from HEK-293 cells) encoding the human adrenergic a1D receptor are used in modified Tris-HCl, pH 7.4 buffer using standard techniques. A 50 μg‡ aliquot of membrane is incubated with 0.6 nM [³H]Prazosin for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 10 μM phentolamine. Membranes are filtered and washed 3 times and the filters are counted to determine [³H]Prazosin 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] Prazosin

Ligand Kd (nM):

0.58

Ligand Concentration:

0.6 nM

Non Specific:

10 µM Phentolamine

Incubation:

60 min at 25°C

Control Inhibitor:

Prazosin

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