Integrity of tritiated orphanin FQ/Nociceptin

Implications for establishing a reliable binding assay

Denise Irene Quigley, Seksiri Arttamangkul, Richard Gordon Allen, David Grandy

Research output: Contribution to journalArticle

5 Citations (Scopus)

Abstract

In the course of establishing a reliable and reproducible binding assay for the orphanin FQ/nociceptin (OFQ/N) ligand-receptor system we used reversed phase-high-performance liquid chromatography (HPLC) (RP-HPLC) to monitor the integrity of [3H]OFQ/N obtained from three different manufacturers. This means of analysis revealed that the stability of [3H]OFQ/N during storage varied considerably depending on the manufacturer. Furthermore, the integrity of [3H]OFQ/N was significantly compromised in the presence of COS-7 cell membranes. Interestingly, if the peptide was added to COS-7 membranes after they had been exposed to low pH it remained intact, suggesting that the peptide's breakdown during binding is, in part, enzymatically mediated. Although a variety of protease inhibitors were tested, none proved completely effective at protecting the tritiated peptide. The intention of the studies presented here was to evaluate OFQ/N binding components, namely the available [3H]OFQ/N ligands, in an effort to standardize the binding conditions for this receptor ligand system. Consequently, this study underscores the importance of monitoring the integrity of the trace ligand being used in a given binding assay. Copyright (C) 2000 Elsevier Science Inc.

Original languageEnglish (US)
Pages (from-to)1111-1118
Number of pages8
JournalPeptides
Volume21
Issue number7
DOIs
StatePublished - Jul 2000

Fingerprint

Assays
Ligands
Peptides
COS Cells
High performance liquid chromatography
Cell membranes
Protease Inhibitors
nociceptin
High Pressure Liquid Chromatography
Cell Membrane
Membranes
Monitoring

Keywords

  • Nociceptin
  • OFQ receptor
  • Orphanin FQ
  • Peptide degradation
  • Radioligand binding

ASJC Scopus subject areas

  • Biochemistry
  • Endocrinology
  • Physiology
  • Cellular and Molecular Neuroscience

Cite this

Integrity of tritiated orphanin FQ/Nociceptin : Implications for establishing a reliable binding assay. / Quigley, Denise Irene; Arttamangkul, Seksiri; Allen, Richard Gordon; Grandy, David.

In: Peptides, Vol. 21, No. 7, 07.2000, p. 1111-1118.

Research output: Contribution to journalArticle

Quigley, Denise Irene ; Arttamangkul, Seksiri ; Allen, Richard Gordon ; Grandy, David. / Integrity of tritiated orphanin FQ/Nociceptin : Implications for establishing a reliable binding assay. In: Peptides. 2000 ; Vol. 21, No. 7. pp. 1111-1118.
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