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The development of a melanocortin MC4 receptor binding assay using the LEADseeker Multimodality Imaging System

ha;-MSH) (Sigma, M-4135)

β-Melanocyte Stimulating Hormone (β-MSH) (Sigma, M-6513)

384-well white flat bottom polystyrene (Corning, 3705)
not treated microplates

Assay buffer: 25 mM HEPES, pH 7.4
5 mM MgCl2
1 mM CaCl2
1 mM 1,10 1,10-phenanthroline monohydrate 0.2% (w/v) protease-free BSA

GraphPad Prism™ software (GraphPad Software)

* Use of the receptor in this application note is not permitted in North America. For further information on the use of this receptor in restricted territories, please contact Euroscreen’s Business Development and Licensing Manager at vlannoy@euroscreen.be


Protocol
Human recombinant melanocortin MC4 receptor membrane preparation produced in CHO-K1 cells was used in conjunction with [125I] NDP-α-MSH (2000 Ci/mmol) and PEI-treated, WGA-coupled SPA Imaging Beads, Type B. Non-specific binding (NSB) was determined in the presence of 2-µM NDP-α-MSH. The standard assay format was as follows:

1. Reagents were added in the following order: assay buffer or buffer containing 2% (v/v) DMSO solution, unlabeled ligand (NSB wells), labeled ligand, premixed bead and membrane*. Total assay volume was 40 µl.

2. Wells contained 10 µl (~ 88 000 dpm) 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 assay buffer in the absence of competing ligand. For competition assays, 10 &mi
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