A3 Human Adenosine GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [¹²⁵I] AB-MECA to human adenosine A3 receptors. CHO-K1 cells stably transfected with a plasmid encoding the human adenosine A3 receptor are used to prepare membranes in modified HEPES pH 7.4 buffer using standard techniques. A 2 μg‡ aliquot of membrane is incubated with 0.5 nM [¹²⁵I] AB-MECA for 60 minutes at 25℃. Non-specific binding is estimated in the presence of 1 μM IB-MECA. Membranes are filtered and washed 3 times and the filters are counted to determine [¹²⁵I] AB-MECA 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] AB-MECA

Ligand Kd (nM):

5.9

Ligand Concentration:

0.5 nM

Non Specific:

1 µM IB-MECA

Incubation:

60 min at 25°C

Control Inhibitor:

IB-MECA

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