High‐Throughput Lectin Microarray‐Based Analysis of Live Cell Surface Glycosylation

Yu Li1, Sheng‐ce Tao2, Heng Zhu3, Jonathan P. Schneck1

1 Department of Pathology, Oncology & Medicine and Institute for Cell Engineering, Johns Hopkins University, Baltimore, Maryland, 2 Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University, Shanghai, China, 3 The High‐Throughput Biology Center, Johns Hopkins University School of Medicine, Baltimore, Maryland
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 12.9
DOI:  10.1002/0471140864.ps1209s63
Online Posting Date:  February, 2011
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Abstract

Lectins, plant‐derived glycan‐binding proteins, have long been used to detect glycans on cell surfaces. However, the techniques used to characterize serum or cells have largely been limited to mass spectrometry, blots, flow cytometry, and immunohistochemistry. While these lectin‐based approaches are well established and they can discriminate a limited number of sugar isomers by concurrently using a limited number of lectins, they are not amenable for adaptation to a high‐throughput platform. Fortunately, given the commercial availability of lectins with a variety of glycan specificities, lectins can be printed on a glass substrate in a microarray format to profile accessible cell‐surface glycans. This method is an inviting alternative for analysis of a broad range of glycans in a high‐throughput fashion and has been demonstrated to be a feasible method of identifying binding‐accessible cell surface glycosylation on living cells. The current unit presents a lectin‐based microarray approach for analyzing cell surface glycosylation in a high‐throughput fashion. Curr. Protoc. Protein Sci. 63:12.9.1‐12.9.7. © 2011 by John Wiley & Sons, Inc.

Keywords: microarrays; lectins; glycosylation; cancer stem cells; immunology; infectious diseases

     
 
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Table of Contents

  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Lyophilized lectins (available from EY Laboratories and Vector Laboratories)
  • Storage buffer (PBS plus 0.02% Tween and 25% glycerol)
  • 2× printing buffer (see recipe)
  • Slide washing solution (see recipe)
  • Slide blocking buffer (see recipe)
  • ≥1×106 cells of interest
  • Phosphate‐buffered saline (PBS; Invitrogen)
  • CFSE cell tracking dye (Invitrogen)
  • Cell binding buffer (see recipe)
  • 384‐well plates (e.g., Nunc Millipore)
  • Schott NexterionR H slides (Schott North America)
  • Microarray printer (e.g., Bio‐Rad ChipWriter Pro)
  • Laboratory marker
  • Nalgene wash bottles or 25‐ml pipets
  • 37°C water bath
  • Adhesive chambered gasket (e.g., Grace Biolabs FlexWell gaskets)
  • Paper towels
  • Microarray slide scanner (e.g., GenePix 4000B)
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Figures

Videos

Literature Cited

Literature Cited
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