5-HT5A 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-HT5A receptors. CHO-K1 cells stably transfected with a plasmid encoding the human serotonin 5-HT5A receptor are used to prepare membranes in modified Tris-HCl pH 7.4 buffer using standard techniques. A 6 μg‡ aliquot of membrane is incubated with 1.7 nM [³H]LSD for 60 minutes at 37℃. Non-specific binding is estimated in the presence of 100 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):

1.8

Ligand Concentration:

1.7 nM

Non Specific:

100 µM Serotonin (5-HT)

Incubation:

60 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