Lectin‐Array Blotting

Raquel Pazos1, Juan Echevarria1, Alvaro Hernandez1, Niels‐Christian Reichardt2

1 Glycotechnology Laboratory, CIC biomaGUNE, San Sebastian, 2 CIBER‐BBN, CIC biomaGUNE, San Sebastian
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 6.12
DOI:  10.1002/cpcb.20
Online Posting Date:  September, 2017
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Abstract

Aberrant protein glycosylation is a hallmark of cancer, infectious diseases, and autoimmune or neurodegenerative disorders. Unlocking the potential of glycans as disease markers will require rapid and unbiased glycoproteomics methods for glycan biomarker discovery. The present method is a facile and rapid protocol for qualitative analysis of protein glycosylation in complex biological mixtures. While traditional lectin arrays only provide an average signal for the glycans in the mixture, which is usually dominated by the most abundant proteins, our method provides individual lectin binding profiles for all proteins separated in the gel electrophoresis step. Proteins do not have to be excised from the gel for subsequent analysis via the lectin array but are transferred by contact diffusion from the gel to a glass slide presenting multiple copies of printed lectin arrays. Fluorescently marked glycoproteins are trapped by the printed lectins via specific carbohydrate‐lectin interactions and after a washing step their binding profile with up to 20 lectin probes is analyzed with a fluorescent scanner. The method produces the equivalent of 20 lectin blots in a single experiment, giving detailed insight into the binding epitopes present in the fractionated proteins. © 2017 by John Wiley & Sons, Inc.

Keywords: electrophoresis; glycosylation; lectins; microarray

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Lectin Array Blotting
  • Support Protocol 1: Lectin Microarray Printing
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Lectin Array Blotting

  Materials
  • Protein samples of interest
  • 300 mM phosphate buffer, pH 8.0 (see recipe)
  • DMSO
  • Alexa Fluor 647 NHS ester (see recipe)
  • 1.0 M Tris·Cl, pH 8.0 (see recipe)
  • 2× Laemmli sample buffer (Bio‐Rad)
  • 2‐Mercaptoethanol (Bio‐Rad)
  • 12% mini‐PROTEAN TGX gel (10‐well, 30 µl, Bio‐Rad)
  • 10× Tris/glycine/SDS electrophoresis buffer (Bio‐Rad)
  • Page Ruler Plus prestained protein ladder (Thermo Scientific)
  • Fixing buffer (see recipe)
  • Phosphate‐buffered saline (PBS) (see recipe)
  • Blocking buffer (see recipe)
  • Microarray printed lectin slides (see protocol 2Support Protocol)
  • Quenching buffer (see recipe)
  • Coomassie blue staining solution (see recipe)
  • Destaining solution (see recipe)
  • Orbital shaker (e.g., Bioblock KL2, Fisher Scientific)
  • Thermomixer comfort (Eppendorf)
  • Mini‐PROTEAN Tetra cell electrophoresis system (Bio‐Rad)
  • Micropipette with gel loader tips
  • PowerPac HC (Bio‐Rad)
  • Plastic containers
  • Filter paper (extra‐thick blot paper, Bio‐Rad, cat. no. 1703966)
  • Glass Coplin staining jars
  • Slide spinner centrifuge (Labnet)
  • Glass plates
  • 1‐kg weight
  • Humidity cabinet (Nüve TK120)
  • Microarray scanner (Agilent G265BA, Agilent Technologies)
  • Plastic spatula
  • Transparent surface
  • ProScanArray Express software (Perkin Elmer)
  • SigmaPlot software
  • VersaDoc imaging system 4000 MP (Bio‐Rad)

Support Protocol 1: Lectin Microarray Printing

  Materials
  • Lectins (EY Laboratories and Vector Laboratories)
  • Reconstitution buffer (see recipe)
  • Printing buffer (see recipe)
  • NHS‐activated hydrogel glass slides (NexterionH, Schott AG)
  • 384‐well plates
  • Ultra‐low volume dispensing robotic systems (e.g., ciFLEXARRAYER S11, Scienion AG)
  • Vacuum sealer
  • Slide containers
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Figures

Videos

Literature Cited

Literature Cited
  Accogli, G., Desantis, S., Martino, N. A., Dell'Aquila, M. E., Gemeiner, P., & Katrlík, J. (2016). A lectin‐based cell microarray approach to analyze the mammalian granulosa cell surface glycosylation profile. Glycoconjugate Journal, 33, 717–724. doi: 10.1007/s10719‐016‐9666‐2.
  Echevarria, J., Royo, F., Pazos, R., Salazar L., Falcon‐Perez, J. M., & Reichardt, N. C. (2014). Microarray‐based identification of lectins for the purification of human urinary extracellular vesicles directly from urine samples. Chembiochem, 15, 1621–1626. doi: 10.1002/cbic.201402058.
  Etxebarria, J., Calvo, J., Martin‐Lomas, M., & Reichardt, N. C. (2012). Lectin‐array blotting: Profiling protein glycosylation in complex mixtures. ACS Chemical Biology, 7, 1729–1737. doi: 10.1021/cb300262x.
  Gupta, G., Surolia, A., & Sampathkumar, S. G. (2010). Lectin microarrays for glycomic analysis. OMICS, 14, 419–436. doi: 10.1089/omi.2009.0150.
  Hirabayashi, J., Kuno, A., & Tateno, H. (2011). Lectin‐based structural glycomics: A practical approach to complex glycans. Electrophoresis, 32, 1118–1128. doi: 10.1002/elps.201000650.
  Marino, K., Bones, J., Kattla, J. J., & Rudd, P. M. (2010). A systematic approach to protein glycosylation analysis: A path through the maze. Nature Chemical Biology, 6, 713–723. doi: 10.1038/nchembio.437.
  Olsen, I. & Wiker, H. G. (1998). Diffusion blotting for rapid production of multiple identical imprints from sodium dodecyl sulfate polyacrylamide gel electrophoresis on a solid support. Journal of Immunological Methods, 220, 77–84. doi: 10.1016/S0022‐1759(98)00147‐1.
  Stevens, J., Blixt, O., James C., Paulson, J. C., & Wilson I. A. (2006). Glycan microarray technologies: Tools to survey host specificity of influenza viruses. Nature Reviews Microbiology, 4, 857–864. doi: 10.1038/nrmicro1530.
  Zhang, L., Luo, S., & Zhang, B. (2016). The use of lectin microarray for assessing glycosylation of therapeutic proteins. MAbs, 8, 524–535. doi: 10.1080/19420862.2016.1149662.
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