Measuring Protein‐Protein and Protein‐Nucleic Acid Interactions by Biolayer Interferometry

Azmiri Sultana1, Jeffrey E. Lee1

1 Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Ontario
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 19.25
DOI:  10.1002/0471140864.ps1925s79
Online Posting Date:  February, 2015
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Abstract

Biolayer interferometry (BLI) is a simple, optical dip‐and‐read system useful for measuring interactions between proteins, peptides, nucleic acids, small molecules, and/or lipids in real time. In BLI, a biomolecular bait is immobilized on a matrix at the tip of a fiber‐optic sensor. The binding between the immobilized ligand and another molecule in an analyte solution produces a change in optical thickness at the tip and results in a wavelength shift proportional to binding. BLI provides direct binding affinities and rates of association and dissociation. This unit describes an efficient approach using streptavidin‐based BLI to analyze DNA‐protein and protein‐protein interactions. A quantitative set of equilibrium binding affinities (Kd) and rates of association and dissociation (ka/kd) can be measured in minutes using nanomole quantities of sample. © 2015 by John Wiley & Sons, Inc.

Keywords: protein‐protein interaction; protein‐DNA interaction; biolayer interferometry; kinetic; binding affinities

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Biolayer Interferometry Kinetics on the BLItz Platform
  • Alternate Protocol 1: Biolayer Interferometry Kinetics on the Octet Platform
  • Support Protocol 1: Preparation of Biomolecules for Streptavidin‐Based BLI Kinetic Experiments
  • Support Protocol 2: Calculation of Required Amounts of Biotin
  • Support Protocol 3: Setup of Controls for BLI Kinetics Experiments
  • Support Protocol 4: Regeneration of Streptavidin BLI Biosensors
  • Reagents and Solutions
  • Commentary
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Biolayer Interferometry Kinetics on the BLItz Platform

  Materials
  • Biotinylated bait (Support Protocols protocol 31 and protocol 42)
  • BLI kinetics buffer (see recipe)
  • Analyte
  • Black 96‐well non‐binding microplate (Greiner, cat no. 655900)
  • 0.5‐ml black microcentrifuge tubes (Argos Technologies, cat no. T7456‐001)
  • Streptavidin‐coated (SA) BLI biosensors (Pall Corp./ForteBio, cat. no. 18‐0009)
  • Biolayer interferometry system: BLItz (Pall Corp./ForteBio)
  • Additional reagents and equipment for setup of controls, testing for nonspecific binding, and optimization of loading density ( protocol 5)

Alternate Protocol 1: Biolayer Interferometry Kinetics on the Octet Platform

  Additional Materials (also see protocol 1)
  • Octet BLI system (Pall Corp./ForteBio)

Support Protocol 1: Preparation of Biomolecules for Streptavidin‐Based BLI Kinetic Experiments

  Materials
  • Materials to the used as bait and analyte samples
  • Phosphate‐buffered saline (PBS; appendix 2E)
  • EZ link sulfo‐NHS‐LC‐biotinylation kit (Thermo Pierce, cat. no. 21335; store at −20°C); see recipe in Reagents and Solutions to prepare stock solution for biotinylation
  • Bradford reagent [Sigma; cat. no. B6916; store at 4°C; also see unit 3.4 (Olsen and Markwell, 2007)]
  • Amicon Ultra‐free concentrators (various molecular weight cutoffs and volumes available; Millipore)
  • Dialysis tubing (FisherBrand; cat. no. 21‐152‐10; store at 4°C) or PD10 gel filtration desalting column (GE Life Science; cat. no. 17‐0851‐01)
  • Additional reagents and equipment for calculating required amount of biotin ( protocol 4), dialysis (Zumstein, ), and Bradford assay for protein concentration (unit 3.4; Olson and Markwell, ) or spectrophotometric determination of protein concentration (unit 3.1; Grimsley and Pace, )

Support Protocol 2: Calculation of Required Amounts of Biotin

  Materials
  • BLI kinetics buffer (see recipe)
  • Biotinylated bait (Support Protocols protocol 31 and protocol 42)
  • Analyte
  • Streptavidin‐coated (SA) BLI biosensors (Pall Corp/ForteBio; cat. no. 18‐0009)
  • 0.5‐ml black microcentrifuge tubes (Argos Technologies; cat no. T7456‐001)
Biolayer interferometry system: BLItz or Octet (Pall Corp/ForteBio)

Support Protocol 3: Setup of Controls for BLI Kinetics Experiments

  Materials
  • Regeneration solution (Table 19.25.2)
  • BLI kinetics buffer (see recipe)
  • Black 96‐well non‐binding microplate (Greiner; cat no. 655900)
  • Used streptavidin‐coated BLI biosensors
  • Biolayer interferometry system: BLItz or Octet (Pall Corp/ForteBio)
  • Additional reagents and equipment for biosensor interferometry measurements ( protocol 1)
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Figures

Videos

Literature Cited

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