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Development of a CRF1 neuropeptide receptor binding assay using LEADseeker Multimodality Imaging System

Key words: CRF1 GPCR receptor binding assay LEADseeker SPA Imaging Beads


In the mammalian brain, the corticotrophin-releasing factor (CRF) system comprises four CRF-related peptides (CRF and urocortin 1, 2, and 3) and two G protein-coupled CRF receptor subtypes (CRF1 and CRF2, which includes CRF2α and CRF2β) (1). These receptor subtypes are positively coupled to adenylyl cyclase and show distinct expression patterns and binding characteristics. CRF1 receptors, for instance, are widely expressed in the brain, the anterior pituitary, and the GI tract (2). Strong evidence suggests that CRF1 receptors play an important role in stress responses and stress-related behaviors mediated through the hypothalamic-pituitary-adrenal (HPA) axis and through extra-hypothalamic systems (3).

This application note describes a 384-well CRF1 neuropeptide receptor binding assay developed using LEADseeker™ Multimodality Imaging System. The miniaturized assay is robust, achieving a Z’ value of 0.64, facilitating its adaptation to automated screening formats.


Materials
Products used

LEADseeker Multimodality Imaging System 18-1140-71

Wheat Germ Agglutinin (WGA) PS SPA RPNQ0260
Imaging Beads

[125I]Tyr0-CRF (ovine) IMQ20237


Other materials required
Human recombinant CRF1 receptor membrane preparation (Euroscreen)

Human urocortin 1 (Bachem)

Astressin (Tocris)


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IHC Polymer Detection Kit (Rabbit DAB)150 slides...
...
Bovine Serum...
Healthy animals 10-32 weeks of age, mixed gender and breed; collected on wet ice following USDA guidelines, using Alsevers solution at 1:2 dilution...
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