Characterization of Nuclear Receptor Ligands by Multiplexed Peptide Interactions

Marie A. Iannone1

1 GlaxoSmithKline, Research Triangle Park, North Carolina
Publication Name:  Current Protocols in Cytometry
Unit Number:  Unit 13.6
DOI:  10.1002/0471142956.cy1306s35
Online Posting Date:  February, 2006
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Abstract

This unit describes a method to evaluate the effect that small molecules have on the binding interactions of a nuclear receptor protein with a series of peptides. The multiplexed microsphere‐based system employs peptide‐coupled microsphere populations that are fluorescently unique and thereby identifiable by flow cytometric analysis. Up to 100 different peptide–nuclear receptor interactions may be analyzed in a single well of a 96‐well microtiter plate. This approach allows rapid and sensitive characterization of nuclear receptor ligands based on nuclear receptor protein–peptide interaction profiles. Since nuclear receptor binding interactions are dynamically related to protein conformation, the approach allows rapid evaluation of nuclear receptor ligands that may impart unique protein structure. The no‐wash format and the high surface density of the microsphere‐coupled interaction partner offer a moderately high‐throughput system to examine low‐ to high‐affinity interactions with excellent sensitivity. This approach, although described for nuclear receptors, may also be applied to other types of molecular interactions.

Keywords: Microsphere; flow cytometry; nuclear receptor; coactivator; corepressor; cofactor; agonist; antagonist; ligand; ligand binding domain; protein conformation

     
 
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Table of Contents

  • Basic Protocol 1: Multiplexed Binding Assay
  • Support Protocol 1: Coupling of Biotinylated Peptides to Lumavidin‐Coated Microspheres
  • Support Protocol 2: Coupling of Biotinylated Nuclear Receptor LBD to Streptavidin‐Alexa fluor 532
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Multiplexed Binding Assay

  Materials
  • Peptide‐coupled microsphere suspension (see protocol 2)
  • PBS‐TBN/DTT (see recipe)
  • DMSO with or without ligand
  • Alexa Fluor 532–labeled NR LBD (see protocol 3)
  • 96‐well flat‐bottom assay plates, non‐binding surface, non‐sterile (Corning)
  • Multichannel pipet
  • LX‐100 flow cytometer with x‐y plate sampler for automated sampling from 96‐well microtiter plates (Luminex Corporation)

Support Protocol 1: Coupling of Biotinylated Peptides to Lumavidin‐Coated Microspheres

  Materials
  • Phosphate buffered saline without calcium or magnesium (PBS; appendix 2A)
  • xMAP LumAvidin‐coated polystyrene microsphere populations (5.6‐µm diameter; Luminex Corporation); store at 4°C in the dark
  • PBS‐TBN/DTT (see recipe)
  • Lyophilized biotinylated peptide (Synpep or American Peptide)
  • DMSO
  • 5 mM D‐biotin (see recipe)
  • 96‐well filter‐bottom plates, MultiScreen BV 1.2‐µm, clear, non‐sterile (Millipore)
  • MultiScreen vacuum manifold for 96‐well plates (Millipore)

Support Protocol 2: Coupling of Biotinylated Nuclear Receptor LBD to Streptavidin‐Alexa fluor 532

  Materials
  • Biotinylated nuclear receptor ligand binding domain (NR LBD; non‐biotinylated receptors are commercially available from Invitrogen, Active Motif, Jena Bioscience, Protein One, as well as other manufacturers)
  • 16 µM streptavidin–Alexa Fluor 532 conjugate (see recipe)
  • 5 mM free D‐biotin (see recipe)
  • PBS‐TBN/DTT (see recipe)
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Figures

Videos

Literature Cited

Literature Cited
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