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Development of an adrenergic α2a receptor binding assay using the LEADseeker Multimodality Imaging System

een, ES-032-M)

Prazosin hydrochloride (Sigma, P7791)

Rauwolscine hydrochloride (Sigma, R104)

Costar™ solid white 384-well microplate (Corning, 3705)

Buffer: 50-mM Tris pH 7.4
5-mM MgCl2
50-mM NaCl

GraphPad Prism™ software v4.0 (GraphPad Software)


Protocol
Human recombinant α2c receptor membrane preparation was used in conjunction with [0-methyl-3H]Rauwolscine ligand and WGA YOx SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 6-µM prazosin. The standard assay format was as follows:

1. Reagents were added in the following order: buffer, unlabeled ligand (NSB wells), labeled ligand, and precoupled bead and membrane. Total assay volume was 50 µl.

2. Diluted membrane and bead were precoupled for 30 min on a roller mixer at 2–8 ºC.

3. Wells contained 10 µl of 6.5-nM [0-methyl3H]Rauwolscine (final concentration 1.3 nM) unless otherwise stated. Wells also contained 5 µg of membrane and 250 µg of WGA YOx SPA Imaging Beads. Precoupled bead and membrane was added in a 20-µl volume.

4. NSB wells contained 10 µl of 30-µM prazosin (final concentration 6 µM) in addition to the above.

5. Plates were sealed and incubated in darkness for 3 h at room temperature (20–25 ºC) unless otherwise stated.

6. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min using quasi-coincident averaging and 3 ×3 binning.


Results
Kd determination
Saturation binding was performed
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