Applications of Lipid Nanodiscs for the Study of Membrane Proteins by Surface Plasmon Resonance

Meg Trahey1, Mavis Jiarong Li1, Hyewon Kwon2, Erica L. Woodahl3, Wynton D. McClary2, William M. Atkins2

1 These authors contributed equally to this work, 2 Department of Medicinal Chemistry, University of Washington, Seattle Washington, 3 Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, Montana
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 29.13
DOI:  10.1002/0471140864.ps2913s81
Online Posting Date:  August, 2015
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Abstract

Methods for the initial steps of surface plasmon resonance analysis of membrane proteins incorporated in lipid nanodiscs are described. Several types of Biacore sensor chips are available and require distinct strategies to immobilize proteonanodiscs on the chip surface. The procedures for immobilization on three of these chips (NTA, antibody coupled CM5, and L1) are described in this unit and results are demonstrated for a model system with cytochrome P4503A4 (CYP3A4) in nanodiscs binding to a polyclonal anti‐CYP3A4 antibody. Advantages and disadvantages of each chip type are considered. © 2015 by John Wiley & Sons, Inc.

Keywords: membrane protein; surface plasmon resonance binding analysis; antibody‐receptor binding; lipid nanodisc; biosensor

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Antibody Binding to Nanodiscs on NTA Chip
  • Basic Protocol 2: Nanodiscs on CM5 Chip Immobilized with Anti‐6‐Histidine Antibody
  • Basic Protocol 3: Nanodiscs on L1 Chip
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Antibody Binding to Nanodiscs on NTA Chip

  Materials
  • Running buffer (see recipe)
  • 350 mM and 3 mM EDTA, pH 8
  • 0.5 mM NiCl 2
  • Purified empty and CYP3A4 nanodiscs (diluted in running buffer, centrifuged 30 min at 16,000 × g, 4°C, to remove particulates)
  • 100 μg/ml affinity‐purified rabbit polyclonal anti‐CYP3A4 antibody in running buffer
  • Series S sensor chip NTA (GE Healthcare Life Sciences)
  • Biacore T200
  • Biacore rubber caps
  • Biacore plastic vials
NOTE: Temperature of chip and sample compartment is set to room temperature (25°C). The running buffer must not contain divalent cations such as Mg2+ or Ca2+.

Basic Protocol 2: Nanodiscs on CM5 Chip Immobilized with Anti‐6‐Histidine Antibody

  Materials
  • HBS‐EP buffer (0.01 M HEPES, pH 7.4, 0.15 M NaCl, 3 mM EDTA, 0.0005% v/v surfactant P20)
  • 0.4 M 1‐ethyl‐3‐(3‐dimethylaminoprolyl)‐carbodiimide (EDC)
  • 0.1 M N‐hydroxysuccinimide (NHS)
  • Mouse anti‐6‐histidine antibody (86.7 μg/ml in 10 mM sodium acetate, pH 4.5)
  • 1.0 M ethanolamine‐HCl, pH 8.5
  • Running buffer (see recipe)
  • 10 mM glycine‐HCl, pH 1.5
  • Purified empty and CYP3A4 nanodiscs (diluted in running buffer, centrifuged 30 min at 16,000 × g, 4°C to remove particulates)
  • 100 μg/ml affinity‐purified rabbit polyclonal anti‐CYP3A4 antibody in running buffer
  • Series S sensor chip CM5 (GE Healthcare Life Sciences)
  • Biacore T200
  • Biacore rubber caps
  • Biacore plastic vials
NOTE: Temperature of chip and sample compartment is set at room temperature (25°C).NOTE: Buffers used for antibody immobilization must not contain amine derivatives (e.g., Tris, sodium azide), which may interfere with the amine coupling process.

Basic Protocol 3: Nanodiscs on L1 Chip

  Materials
  • Running buffer (see recipe)
  • 20 mM CHAPS in running buffer
  • Purified empty and CYP3A4 nanodiscs (diluted in running buffer, centrifuged 30 min at 16,000 × g, 4°C, to remove particulates)
  • 0.2 mg/ml BSA in running buffer
  • 100 μg/ml affinity‐purified rabbit polyclonal anti‐CYP3A4 antibody in running buffer
  • Series S Sensor Chip L1 (GE Healthcare Life Sciences)
  • Biacore T200
  • Biacore rubber caps
  • Biacore plastic vials
NOTE: Temperature of the chip and sample compartment is set at room temperature (25°C).
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Figures

Videos

Literature Cited

Literature Cited
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