MC3 Human Melanocortin GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures the binding of [¹²⁵I]NDP-a-MSH to human melanocortin MC3 receptors. HEK-293 cells stably transfected with a plasmid encoding the human melanocortin-MC3 receptor are used to prepare membranes in modified HEPES-KOH buffer at pH 7.0 using standard techniques. A 3 μg‡ aliquot is incubated with 0.035 nM [¹²⁵I]NDP-a-MSH for 60 minutes at 37°C. Non-specific binding is estimated in the presence of 3 μM NDP-a-MSH. Membranes are filtered and washed 3 times and filters are counted to determine [¹²⁵I]NDP-aMSH 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:

[¹²⁵I] NDP-alpha-MSH

Ligand Kd (nM):

0.24

Ligand Concentration:

0.035 nM

Non Specific:

3 µM NDP-alpha-MSH

Incubation:

60 min at 37°C

Control Inhibitor:

NDP-alpha-MSH

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