RARalpha Human Retinoic Acid NHR Binding (Agonist Radioligand) Assay, Panlabs

Procedure Summary:

Human recombinant RARα-LBD expressed in insect cells are used in modified Tris-HCl buffer pH 7.4. A 0.025 μg‡ aliquot is incubated with 3 nM [³H]9-cis-Retinoic acid and 1 μg Anti-GST antibody for 2 hours at 4°C. Non-specific binding is estimated in the presence of 1 μM 9-cis-retinoic acid. Receptors are counted to determine [³H]9-cis-Retinoic acid specifically bound. Compounds are screened at 10 μM.

Ligand:

[³H] 9-cis-Retinoic acid

Ligand Kd (nM):

3.8

Ligand Concentration:

3 nM

Non Specific:

1 µM 9-cis-Retinoic acid

Incubation:

120 min at 4°C

Control Inhibitor:

9-cis-Retinoic acid

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