5-HT6 Human Serotonin GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H]Lysergic acid diethylamide (LSD) to human serotonin 5-HT6 receptors. HeLa/E6-7 cells stably transfected with a plasmid encoding the human serotonin 5-HT6 receptor are used to prepare membranes in modified Tris-HCl pH 7.4 buffer using standard techniques. A 30 μg‡ aliquot of membrane is incubated with 1.5 nM [³H]LSD for 120 minutes at 37℃. Non-specific binding is estimated in the presence of 5 µM 5-HT. Membranes are filtered and washed 3 times and the filters are counted to determine [³H]LSD 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] Lysergic acid diethylamide (LSD)

Ligand Kd (nM):

1.3

Ligand Concentration:

1.5 nM

Non Specific:

5 µM Serotonin (5-HT)

Incubation:

120 min at 37°C

Control Inhibitor:

Methiothepin

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