( with dilutions of [0-methyl- 3 H]Rauwo...)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 K_d 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 K_i 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 K_i 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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