cro;l of competing ligand prepared in assay buffer containing 2% (v/v) DMSO was added with [125
I] NDP-α-MSH (10 µl, as above), precoupled bead and receptor (20 µl), also giving a total assay volume of 40 µl.
3. NSB wells contained 10 µl of 2-nM [125I] NDP-α-MSH (final concentration 0.5 nM), 31.25 µg of SPA Imaging Bead, 250 ng of receptor preparation added together in a 20 µl volume, and 10 µl of 8-µM unlabeled NDP-α-MSH (final concentration 2 µM).
4. For DMSO tolerance studies, DMSO was diluted in assay buffer to give final concentrations per well as shown in the results.
5. Plates were sealed and incubated overnight at room temperature (20–25 ºC).
6. Following incubation, plates were imaged on the LEADseeker Multimodality Imaging System for 5 min with quasi-coincident averaging and 3 ×3 binning.
7. For background estimation, bead-only wells (31.25 µg of bead/well, 20 µl), plus assay buffer (20 µl) were included in all experiments. Typically backgrounds exhibited from bead-only wells were less than 10 integrated optical density (IOD) units, and were routinely subtracted from total and NSB values during data analyses.
* Diluted membrane and bead were precoupled at room temperature immediately prior to assay addition.
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Saturation binding was carried out with dilutions of [125I] NDP-α-MSH to give a range of concentrations from 4.05 pM to 10.43 nM in the assay wells. Figure 1 shows the saturation curve which was fitted using non-linear regression with the data analysis package GraphPad Prism v4.0. A Kd value of 0.931 nM (95% confidence intervals 0.744 to 1.117 nM) was estimated directly from the curve
. The development of a CXCR2 chemokine receptorbinding assay using the LEADseeker Multimodality Imaging System2
. The development of a melanocortin MC5 receptor binding assay using the LEADseeker Multimodality Imaging System3
. The development of a calcitonin-gene related peptide (CGRP) receptor-binding assay using the LEADseeker Multimodality Imaging System4
. The development of a somatostatin sst4 receptor-binding assay using the LEADseeker Multimodality Imaging System5
. The development of a serotonin 5-HT2C receptor-binding assay using the LEADseeker Multimodality Imaging System6
. Screening for potential beta 2-adrenergic receptor antagonists using CypHer5E and IN Cell Analyzer 10007
. The use of CypHer5 for receptor internalization studies in both a range of GPCRs and a non-GPCR8
. The use of CypHer5E and the IN Cell Analyzer 1000 for live-cell receptor internalization studies9
. Screening for β2-adrenergic receptor agonists using the pH-sensitive dye,CypHer5, and the IN Cell Analyzer 300010
. Development of a histamine H1 receptor binding assay using the LEADseeker Multimodality Imaging System11
. Development of a sensitive enzyme fragment complementation assay for cyclic adenosine 3, 5 monophosphate and its validation for pharmacological screening at G protein-coupled receptors