Advances in Lectin Microarray Technology: Optimized Protocols for Piezoelectric Print Conditions

Kanoelani T. Pilobello1, Praveen Agrawal1, Richard Rouse2, Lara K. Mahal1

1 Department of Chemistry, Biomedical Chemistry Institute, New York University, New York, 2 HTS Resources, San Diego, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch120035
Online Posting Date:  March, 2013
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Abstract

Lectin microarray technology has been used to profile the glycosylation of a multitude of biological and clinical samples, leading to new clinical biomarkers and advances in glycobiology. Lectin microarrays, which include >90 plant lectins, recombinant lectins, and selected antibodies, are used to profile N‐linked, O‐linked, and glycolipid glycans. The specificity and depth of glycan profiling depends upon the carbohydrate‐binding proteins arrayed. The current set targets mammalian carbohydrates including fucose, high mannose, branched and complex N‐linked, α‐ and β‐galactose and GalNAc, α‐2,3‐ and α‐2,6‐sialic acid, LacNAc, and Lewis X epitopes. Previous protocols have described the use of a contact microarray printer for lectin microarray production. Here, an updated protocol that uses a non‐contact, piezoelectric printer, which leads to increased lectin activity on the array, is presented. Optimization of print and sample hybridization conditions and methods of analysis are discussed. Curr. Protoc. Chem. Biol. 5:1‐23 © 2013 by John Wiley & Sons, Inc.

Keywords: carbohydrate analysis; glycomics; lectin microarray; Nano‐Plotter; piezoelectric

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Lectin Microarray Print Preparation and General Printing Protocol Using Non‐Contact Printer (Nano‐Plotter)
  • Basic Protocol 2: Sample Preparation, Labeling, Hybridization, and Data Analysis
  • Alternate Protocol 1: Using an Advanced Program Interface (Multitask) for Customizable Automation
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Lectin Microarray Print Preparation and General Printing Protocol Using Non‐Contact Printer (Nano‐Plotter)

  Materials
  • Nexterion H slides (Schott): store ∼1 year at −20°C in a vacuum‐tight packet
  • Lectins (see Table 12.0.3500)
  • Monosaccharides: 99.5+% α‐D‐lactose monohydrate, 99+% D‐(+)‐mannose, 99+% D‐(+)‐galactose, 97% l‐(–)‐fucose, N acetyl‐glucosamine (Acros Organics)
  • PBS‐Tween (PBS‐T): 0.1 M NaH 2PO 4, 0.15 M NaCl, 0.01% Tween‐20, pH 7.2
  • 384‐well plates
  • Centrifuge with microplate adaptors
  • Nano‐Plotter 2.1 piezoelectric printer (GeSiM) with cooled microwell plate holder and cooled printing deck (Figure )

Basic Protocol 2: Sample Preparation, Labeling, Hybridization, and Data Analysis

  Materials
  • Glycoproteins (Sigma) or cells (harvested from tissue culture)
  • NHS‐Cy3 or ‐Cy5 dye (GE Healthcare Life Sciences; store at 4°C in the dark)
  • Cy dye labeling buffer: 0.1 M Na 2CO 3 in H 2O, pH 9.3, prepare fresh
  • 250 mM Tris buffer, pH 6.8
  • Phosphate‐buffered saline (PBS): 0.1 M NaH 2PO 4, 0.15 M NaCl, pH 7.3
  • Lectin array (see protocol 1; printed slides should be stored under vacuum up to 1 month at −20°C)
  • Blocking solution: 25 mM ethanolamine in 100 mM sodium borate, pH 8.0
  • PBS‐Tween (PBS‐T) wash buffer: 0.1 M NaH 2PO 4, 0.15 M NaCl, 0.01% Tween 20, pH 7.2
  • Labotron shaker AK15/6 (Bottmingen)
  • VialTweeter sonicator (Heischler)
  • Ultracentrifuge (Optima L‐100K ultracentrifuge, Beckman Coulter)
  • 25‐G needles and 1‐ml syringes
  • Coplin jars
  • Slide spinner (model C1303; Labnet International)
  • 24‐well multi‐well hybridization cassette (FAST frame; Arrayit)
  • GenePix 4300A fluorescent slide scanner (Molecular Devices)
  • Genepix Pro 7 software (Molecular Devices)
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Figures

Videos

Literature Cited

Literature Cited
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Supplementary Material