GABAB (B1/B2) Rat GABAB GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Whole brains of male Wistar derived rats weighing 175 ± 25 g are used to prepare GABAB receptors in modified Tris-HCl buffer pH 7.4. A 5 mg‡ aliquot is incubated with 0.6 nM [³H]CGP-54626 for 20 minutes at 25℃. Non-specific binding is estimated in the presence of 100 μM CGP-54626. Membranes are filtered and washed, the filters are then counted to determine [³H]CGP-54626 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] CGP-54626

Ligand Kd (nM):

2.3

Ligand Concentration:

0.6 nM

Non Specific:

100 µM CGP-54626

Incubation:

20 min at 25°C

Control Inhibitor:

Cgp-54626

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