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

with dilutions of [0-methyl-3H]Rauwolscine to give a range of concentrations from 0.07 to 8.8 nM in the wells. Plates were incubated for 5 h prior to imaging. Figure 1 shows the saturation curve, which was fitted using nonlinear regression with the data analysis software package GraphPad Prism v4.0. Over two experiments the mean Kd value was determined to be 1.3 nM (95% confidence intervals 0.8 to 1.9 nM), estimated directly from the curve. Figure 1 is representative of the data obtained.

DMSO tolerance
Dilutions of DMSO were prepared in assay buffer to give a range of concentrations from 0.25% (v/v) to 5% (v/v) in the well. The results shown in Figure 2 indicate the assay was tolerant to DMSO up to a final concentration of 1% (v/v).

IC50 determination
Competitive binding of 1.3-nM [0-methyl-3H]Rauwolscine with rauwolscine (Fig 3A) and prazosin (Fig 3B) was assessed and the IC50 value of each ligand calculated. Final concentrations in the wells were from 0.0004 to 4000 nM and 0.25 to 20000 nM respectively. DMSO was present at a final concentration of 1% v/v in each well.

Over two experiments, the mean rauwolscine IC50 value was determined to be 3.1 nM (95% confidence interval range 2.7–3.7 nM) and the mean Ki value was 1.6 nM (95% confidence intervals 1.3–1.8 nM). The mean prazosin IC50 value was determined to be 430 nM (95% confidence interval range 336–535 nM) and the mean Ki value was 215 nM (95% confidence intervals 168–267 nM).

Time course
A time course was performed using standard reagent concentrations detailed in the protocol with an increased incubation time of 17 h. The assay a
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