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Development of Radioligand Binding Assays for the Motilin Receptor Using ScreenReady Targets.

fly, 12.5 μl of binding buffer without (total binding determination) or with motilin at 1 μM final concentration (non-specific binding determination) were first added to membrane-coated FlashPlate microplates, followed by the addition of 12.5 μl [125I]-motilin. Incubation was performed at room temperature for 1 to 5 hours before counting on a PerkinElmer TopCount instrument (30 seconds/well).

Kd and Ki values were calculated using GraphPad Prism Software. Signal to background ratio (S/B) (total values/ nonspecific values) was calculated using a concentration of radioligand close to the Kd value.

Results and Discussion

Saturation Binding Assays
Membranes from CHO cells expressing the human motilin MTL-R1A receptor were immobilized into 96-well FlashPlate microplates and the affinity of [125I]-motilin for the receptor was determined. Saturation experiments using motilin ScreenReady Targets were performed on the day of membrane immobilization (Figure 2, A) or upon storage at 4C for three months (Figure 2, B). A similar single class of high affinity binding sites was detected from the saturation experiment performed on the day of membrane immobilization or after three months upon storage at 4C (Kd = 1.0 nM and 0.75 nM, respectively). Calculated Kd values were comparable to those previously reported (Feighner et al ., 1999; Thielemans et al ., 2001). Bmax values were also very similar in both assays (4.3 pmol/mg at t= 0 and 3.6 pmol/mg at t= 3 months). S/B values calculated at a concentration of radioligand close to the Kd value were comparable in both assays (5.1 vs. 4.6), showing that the immobilized motilin receptor maintained its pharmacological binding properties for at least three months at 4C.

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