( Costar™ solid white 384-wel...)Development of a CRF1 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System

Costar™ solid white 384-well NBS™ coated microplate (Corning)

Protease-free BSA (Fluka)

EDTA-free protease inhibitor cocktail tablets (Roche)

Buffer: 25 mM Hepes pH 7.4
5 mM MgCl₂
1 mM CaCl₂
0.2% BSA (w/v) plus protease inhibitor cocktail

GraphPad Prism™ software v4.0 (GraphPad Software)

Protocol
Human recombinant CRF1 receptor membrane preparation was used in conjunction with [¹²⁵I]Tyr⁰-CRF ligand and WGA PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 200 nM urocortin. The standard assay format was as follows:

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

2) Wells contained 10 µl of 1.5 nM [¹²⁵I]Tyr⁰-CRF (final concentration 0.3 nM) unless otherwise stated. Wells also contained 0.3 µg of membrane and 30 µg of beads, which were added in 10 µl volumes.

3) NSB wells contained 10 µl of 1000 nM urocortin (final concentration 200 nM) in addition to the above.

4) Plates were sealed and incubated in darkness for 18 h at room temperature (20–25 °C).

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

Results
K_d determination
Saturation binding was performed with dilutions of [¹²⁵I]Tyr⁰-CRF to give a range of concentrations from 0.06 to 4.96 nM in the wells. Figure 1 shows the saturation curve, which was fitted using non-linear regression with the data analysis s
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