B2 Human Bradykinin GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

Human recombinant bradykinin B2 receptors expressed in Chem-1 cells are used in modified HEPES buffer pH 7.4. A 5 μg‡ aliquot is incubated with 0.5 nM [³H]Bradykinin for 60 minutes at 25°C. Non-specific binding is estimated in the presence of 5 μM bradykinin. Receptors are filtered and washed, the filters are then counted to determine [³H]Bradykinin 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] Bradykinin

Ligand Kd (nM):

0.85

Ligand Concentration:

0.5 nM

Non Specific:

5 µM Bradykinin

Incubation:

60 min at 25°C

Control Inhibitor:

Bradykinin

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