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Preparation and Analysis of Glycan Microarrays

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Abstract
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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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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

GO TO THE FULL PROTOCOL: PDF or HTML at Wiley Online Library 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 2 O

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 2 O

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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Figure 12.10.1 Preparation of natural glycan microarrays. After isolating the glycans from a natural source, they are labeled with a bifunctional fluorescent dye (e.g., AEAB), captured on glass slides, and interrogated by glycan‐binding proteins (GBPs).

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Figure 12.10.2 Schematic of natural, defined glycan array printed with 14 identical subarrays per slide. A multi‐chamber adapter can be placed over the slide surface to create 14 individual chambers for 14 different, simultaneous assays. In this example, each of the 40 glycans is printed in replicates of 4. In some cases, the printed compounds include controls such as glycopeptides and biotin.

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Figure 12.10.3 Con A binding to the CFG glycan array. The top panel is an image of fluorescently detected Con A binding to the glycan array slide. The replicates of one set of 6 spots, corresponding to one glycan, are noted. The bottom panel is a histogram of quantified Con A binding to the glycan array, where the x ‐axis is the chart number (glycan number) and the y ‐axis is relative fluorescent units (RFU).

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Figure 12.10.4 The binding of biotinylated SNA to a natural glycan array containing 52 GAEABs and four controls. After obtaining a fluorescent image of the biotinylated SNA bound by cyanine5‐streptavidin, binding is quantified and displayed as a histogram of the average RFU bound to each glycan. The eight glycan structures that were bound by SNA are displayed next to their respective peak.

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Literature Cited

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