A2A Human Adenosine GPCR Binding Agonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H]CGS-21680 to human adenosine A2A receptors. HEK-293 (human embryonic kidney) cells stably transfected with a plasmid encoding the human adenosine A2A receptor are used to prepare membranes in modified Tris-HCl pH 7.4 buffer using standard techniques. A 15 μg‡ aliquot of membrane is incubated with 50 nM [³H]CGS-21680 for 90 minutes at 25℃. Non-specific binding is estimated in the presence of 50 µM NECA. Membranes are filtered and washed 3 times and the filters are counted to determine [³H]CGS-21680 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] CGS-21680

Ligand Kd (nM):

64

Ligand Concentration:

0.05 µM

Non Specific:

50 µM NECA

Incubation:

90 min at 25°C

Control Inhibitor:

CGS 21680

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