Preparation and Analysis of Glycan Microarrays

Jamie Heimburg‐Molinaro1, Xuezheng Song1, David F. Smith1, Richard D. Cummings1

1 The Glycomics Center at Emory University School of Medicine, Atlanta, Georgia
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.10
DOI:  10.1002/0471140864.ps1210s64
Online Posting Date:  April, 2011
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Abstract

Determination of the binding specificity of glycan‐binding proteins (GBPs), such as lectins, antibodies, and receptors, has traditionally been difficult and laborious. The advent of glycan microarrays has revolutionized the field of glycobiology by allowing simultaneous screening of a GBP for interactions with a large set of glycans in a single format. This unit describes the theory and method for production of two types of glycan microarrays (chemo/enzymatically synthesized and naturally derived), and their application to functional glycomics to explore glycan recognition by GBPs. These procedures are amenable to various types of arrays and a wide range of GBP samples. Curr. Protoc. Protein Sci. 64:12.10.1‐12.10.29. © 2011 by John Wiley & Sons, Inc.

Keywords: glycan microarrays; natural glycan microarrays; glycan‐binding specificities; Consortium for Functional Glycomics

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: The Binding Specificity of Biotinylated Plant Lectins Detected with Fluorescent Streptavidin
  • Alternate Protocol 1: Detecting the Glycan Epitopes for Mouse Monoclonal Antibodies Using Fluorescent Anti‐Mouse IgG
  • Alternate Protocol 2: Specificity of Directly Labeled Influenza Virus Binding to Sialylated Glycans
  • Generation of Natural Glycan Arrays
  • Basic Protocol 2: Preparation of a Natural Glycan Microarray
  • Basic Protocol 3: Binding Assay for Natural, Defined Glycan Microarray
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: The Binding Specificity of Biotinylated Plant Lectins Detected with Fluorescent Streptavidin

  Materials
  • Washing buffers (e.g., TSM, TSMWB, TSMBB; see reciperecipes)
  • TSM binding buffer (TSMBB; see recipe)
  • Biotinylated lectin (commercially available, e.g., Vector Labs)
  • Glycan printed slides, printed on one side of the slide (available from the CFG by request)
  • TSM wash buffer (TSMWB; see recipe)
  • Alexa Fluor‐488‐streptavidin (Invitrogen)
  • dH 2O
  • 100‐ml Coplin jars for washing slides
  • Coverslips (Fisher Scientific, cat. no. 12‐545F)
  • Humidified slide‐processing chambers (Fisher Scientific, cat. no. NC9091416) or homemade system using a petri dish with wet paper towels at the bottom of the chamber
  • Slide spinner centrifuge (Labnet, cat. no. C1301)
  • ProScanArray Scanner (Perkin Elmer)
  • ScanArray quantitation software (Scanarray Express, Perkin Elmer or Imagene, Biodiscovery)

Alternate Protocol 1: Detecting the Glycan Epitopes for Mouse Monoclonal Antibodies Using Fluorescent Anti‐Mouse IgG

  • Mouse monoclonal IgG antibody
  • Cyanine 5‐streptavidin (Invitrogen, cat. no. 43‐4316)
  • Appropriate secondary antibody to detect the monoclonal antibody being tested, fluorescently labeled if available (e.g., Alexa Fluor‐488‐labeled anti–mouse IgG)

Alternate Protocol 2: Specificity of Directly Labeled Influenza Virus Binding to Sialylated Glycans

  • Fluorescently labeled virus
  • Cyanine 5‐ (Invitrogen, cat. no. 43‐4316) or Alexa Flour 488‐streptavidin

Basic Protocol 2: Preparation of a Natural Glycan Microarray

  Materials
  • 2‐(N‐aminoethyl)amino benzamide (AEAB) (Song et al., )
  • Dimethyl sulfoxide (DMSO; Fisher Scientific, ACS grade)
  • Acetic acid (AcOH; Fisher Scientific, ACS grade)
  • Sodium cyanoborohydride (NaCNBH 3; Sigma‐Aldrich, 95%)
  • Free reducing glycans (lyophilized; e.g., Sigma)
  • Acetonitrile (HPLC grade; Fisher Scientific )
  • Trifluoroacetic acid (TFA; HPLC grade; Fisher Scientific)
  • Lactose‐AEAB
  • 3,5‐dihydroxylbenzoic acid (DHB; Sigma‐Aldrich, 98%)
  • 2× Printing buffer: 0.6 M sodium phosphate, pH 8.5
  • Blocking buffer: 50 mM ethanolamine in 0.1 M Tris buffer, pH 9.0
  • MilliQ water
  • Vortex mixer
  • 2‐ml microcentrifuge tubes
  • 65°C heat block
  • SpeedVac
  • Shimadzu HPLC system
  • Porous graphitized carbon (PGC) HPLC column (Thermo, 4.6 × 150–mm) with a Javelin cartridge (4 × 20–mm)
  • SPD‐20A UV/vis dual λ detector
  • RF‐10xl fluorescence detector
  • Ultraflex II MALDI‐TOF/TOF (Bruker)
  • Labconco centra‐vap concentrator
  • 384‐well source plate with V‐shape wells (Bio‐Rad, cat. no. MSP3842)
  • Piezorray printer (Perkin‐Elmer)
  • Nexterion NHS‐activated microarray slides (Schott, Slide H)
  • Vacuum desiccator
  • Water bath at 55°C

Basic Protocol 3: Binding Assay for Natural, Defined Glycan Microarray

  Materials
  • TSM (see recipe)
  • TSM washing buffer (TSMWB; see recipe)
  • TSM binding buffer (TSMBB; see recipe)
  • Biotinylated lectin (commercially available, e.g., Vector Labs)
  • Natural, defined glycan array printed slides, printed on one side of the slide, 14 identical subarrays per slide (e.g., protocol 4)
  • Streptavidin‐Alexa Fluor‐488 (Invitrogen)
  • dH 2O
  • Desiccator, room temperature
  • Multi‐chamber adapter (e.g., Grace Biolabs)
  • Rotating shaker
  • Vacuum suction with clean pipet tips
  • Parafilm or plastic adhesive cover
  • 100‐ml Coplin jar for washing slides
  • Slide centrifuge
  • ProScanArray Scanner (Perkin Elmer)
  • ScanArray quantitation software (Scanarray Express, Perkin Elmer or Imagene, Biodiscovery)
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Figures

Videos

Literature Cited

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