A1 Human Adenosine GPCR Binding Antagonist Radioligand LeadHunter Assay - TW

Procedure Summary:

This assay measures binding of [³H]DPCPX to adenosine A1 receptors. CHO-K1 cells stably transfected with a plasmid encoding the human adenosine A1 receptor are used to prepare membranes in modified HEPES pH 7.4 using standard techniques. A 10 μg‡ aliquot of membrane is incubated with 1 nM [³H]DPCPX for 90 minutes at 25°C. Non-specific binding is estimated in the presence of 100 µM R(-)-PIA. Membranes are filtered and washed 3 times and the filters are counted to determine [³H]DPCPX 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] DPCPX

Ligand Kd (nM):

1.4

Ligand Concentration:

1 nM

Non Specific:

100 µM R(-)-PIA

Incubation:

90 min at 25°C

Control Inhibitor:

R(-)-PIA

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