( PA Imaging Beads P ...)Development of an NK2 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System

(2-[¹²⁵I]iodohistidyl¹) Neurokinin A ([¹²⁵I]Substance K) IM168

Other materials required
Human recombinant NK2 receptor membrane preparation (Euroscreen)

[β-Ala⁸]-Neurokinin A fragment (4–10) (Sigma)

Costar™ solid white 384-well microplate (Corning)

Protease-free BSA (Sigma)

Buffer: 50 mM Tris pH 7.4
5 mM MgCl₂
2 mM EDTA
0.2% BSA (w/v)

GraphPad Prism™ software v4.0 (GraphPad Software)

Protocol
Human recombinant NK2 receptor membrane preparation was used in conjunction with [¹²⁵I]NKA ligand and WGA-PEI Type A PS SPA Imaging Beads. Non-specific binding (NSB) was determined in the presence of 5 µM NKA (4–10). The standard assay format was as follows:

1) Reagents were added in the following order: buffer, unlabeled ligand (NSB wells), labeled ligand, membrane, and bead. Total assay volume was 50 µl.

2) Each well contained 10 µl of 0.85 nM [¹²⁵I]NKA (final concentration 0.17 nM) unless otherwise stated, 2.5 µg of membrane, and 250 µg of beads. Membrane and beads were added in 10 µl volumes.

3) NSB wells contained 20 µl of 13 µM NKA (4–10) (final concentration 5 µM) in addition to the above.

4) Plates were sealed and incubated in darkness for 5 h at room temperature (20–25 °C).

5) Following incubation, plates were imaged on LEADseeker Multimodality Imaging System for 5 min using quasi-coincident averaging and 3 ×3 binning.

Results
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