CGRP (CLR/RAMP1) Human Calcitonin GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant CGRP1 calcitonin receptors expressed in Chem-1 cells are used in modified HEPES buffer pH 7.4. A 0.8 μg‡ aliquot is incubated with 30 pM [¹²⁵I]CGRP for 90 minutes at 25°C. Non-specific binding is estimated in the presence of 1 μM α-CGRP (human). Membranes are filtered and washed, the filters are then counted to determine [¹²⁵I]CGRP 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] CGRP (human)

Ligand Kd (nM):

0.11

Ligand Concentration:

0.03 nM

Non Specific:

1 µM alpha-CGRP (human)

Incubation:

90 min at 25°C

Control Inhibitor:

alpha-CGRP (human)

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