Using Single Lectins to Enrich Glycoproteins in Conditioned Media

Manveen K. Sethi1, Susan Fanayan2

1 Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, 2 Australian School of Advanced Medicine, Macquarie University, Sydney
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 24.6
DOI:  10.1002/0471140864.ps2406s81
Online Posting Date:  August, 2015
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Abstract

Lectins are sugar‐binding proteins that can recognize and bind to carbohydrates conjugated to proteins and lipids. Coupled with mass spectrometry technologies, lectin affinity chromatography is becoming a popular approach for identification and quantification of glycoproteins in complex samples such as blood, tumor tissues, and cell lines. Given the commercial availability of a large number of lectins that recognize diverse sugar structures, it is now possible to isolate and study glycoproteins for biological and medical research. This unit provides a general guide to single‐lectin‐based enrichment of glycoproteins from serum‐free conditioned media. Due to the unique carbohydrate specificity of most lectins and the complexity of the samples, optimization steps may be required to evaluate different elution buffers and methods as well as binding conditions, for each lectin, for optimal recovery of bound glycoproteins. © 2015 by John Wiley & Sons, Inc.

Keywords: lectin; conditioned media; glycoprotein enrichment; cell line

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Enrichment of Glycoproteins Using Agarose Bound Lectin Affinity Chromatography
  • Support Protocol 1: Preparation of Concentrated Serum‐Free Conditioned Media
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
  • Tables
     
 
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Materials

Basic Protocol 1: Enrichment of Glycoproteins Using Agarose Bound Lectin Affinity Chromatography

  Materials
  • Agarose‐bound lectin (Vector Laboratories, Sigma Aldrich, or Life Technologies)
  • Binding buffer (see recipe)
  • Concentrated conditioned serum‐free medium (see protocol 2Support Protocol)
  • CHAPS detergent (Sigma‐Aldrich, cat. no. C3023)
  • Elution buffer, lectin‐specific (see Table 24.6.1)
  • 25 mM Tris·Cl, pH 7.5 ( appendix 2E)
  • Poly‐prep chromatography columns with porous discs (Bio‐Rad Laboratories, cat. no. 731‐1550), empty polypropylene gravity flow columns with 2‐ml bed volume and 10‐ml sample reservoir
  • 15‐ml conical tubes
  • Amicon Ultra‐15 10K centrifugal filter units (Millipore, cat. no. UFC901008)
  • SpeedVac
  • Additional reagents and equipment for determination of protein concentration (unit 3.4; Olson and Markwell, )

Support Protocol 1: Preparation of Concentrated Serum‐Free Conditioned Media

  Materials
  • Cells (e.g., LIM2405 colon cancer cells)
  • Cell culture medium (e.g., RPMI 1640 available from Invitrogen or Sigma‐Aldrich)
  • Fetal bovine serum (FBS; available from Invitrogen or Sigma‐Aldrich)
  • Media supplements (e.g., human recombinant insulin and glutamine; available from Invitrogen or Sigma‐Aldrich)
  • Phosphate‐buffered saline (PBS), pH 7.2 ( appendix 2E)
  • 100‐mm culture dish
  • Amicon Ultra‐15 10K centrifugal filter units (Millipore, cat. no. UFC901008)
  • Additional reagents and equipment for determination of protein concentration (unit 3.4; Olson and Markwell, )
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Figures

Videos

Literature Cited

Literature Cited
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