alpha2 (Non-Selective) Rat Adrenoceptor GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Cerebral cortices of male Wistar derived rats weighing 175 ± 25 g are used to prepare adrenergic a2 receptors in modified HEPES buffer pH 7.4. A 7.5 mg‡ aliquot is incubated with 0.7 nM [³H]Rauwolscine for 30 minutes at 25℃. Non-specific binding is estimated in the presence of 1 µM yohimbine. Membranes are filtered and washed 3 times and the 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):

7.8

Ligand Concentration:

0.7 nM

Non Specific:

1 µM Yohimbine

Incubation:

30 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