5-HT2C Human Serotonin GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H]Mesulergine to human serotonin 5-HT2C receptors. CHO-K1 cells stably transfected with a plasmid encoding the human serotonin 5-HT2C receptor are used to prepare membranes in modified Tris-HCl pH 7.4 buffer using standard techniques. A 3.2 μg‡ aliquot of membrane protein is incubated with 1.0 nM [³H]Mesulergine for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 1 μM mianserin. Membranes are filtered and washed 3 times and the filters are counted to determine [³H]Mesulergine 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] Mesulergine

Ligand Kd (nM):

1.1

Ligand Concentration:

1 nM

Non Specific:

1 µM Mianserin

Incubation:

60 min at 25°C

Control Inhibitor:

RS 102221

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