Using Antibody Arrays to Measure Protein Abundance and Glycosylation: Considerations for Optimal Performance

Brian B. Haab1, Katie Partyka1, Zheng Cao1

1 Van Andel Research Institute, Grand Rapids, Michigan
Publication Name:  Current Protocols in Protein Science
Unit Number:  Unit 27.6
DOI:  10.1002/0471140864.ps2706s73
Online Posting Date:  September, 2013
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Abstract

Antibody arrays provide a valuable method for obtaining multiple protein measurements from small volumes of biological samples. Antibody arrays can be designed to target not only core protein abundances (relative or absolute abundances, depending on the availability of standards for calibration), but also posttranslational modifications, provided antibodies or other affinity reagents are available to detect them. Glycosylation is a common modification that has important and diverse functions in both normal and disease biology. Significant progress has been made in developing methods for measuring glycan levels on multiple specific proteins using antibody arrays and glycan‐binding reagents. This unit describes practical approaches for developing, optimizing, and using antibody array assays to determine both protein abundance and glycosylation state. Low‐volume arrays can be used to reduce sample consumption, and a new way to improve the binding strength of particular glycan‐binding reagents through multimerization is discussed. These methods can be useful for a wide range of biological studies in which glycosylation may change and/or affect protein function. Curr. Protoc. Protein Sci. 73:27.6.1‐27.6.16. © 2013 by John Wiley & Sons, Inc.

Keywords: antibody arrays; glycosylation; lectins

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Array‐Based Sandwich Assays for Protein Detection and Quantification
  • Basic Protocol 2: Detecting Glycans on Proteins Captured by Antibody Arrays
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Array‐Based Sandwich Assays for Protein Detection and Quantification

  Materials
  • Sample to be tested
  • 1× phosphate‐buffered saline (PBS, appendix 2E), pH 7.4
  • 2× sample dilution buffer (see recipe)
  • Capture antibody arrays prepared on microscope slides coated with nitrocellulose or N‐hydroxysuccinimide (NHS) hydrophilic polymer (several commercial coated slides are available), optionally with defined wells (see Strategic Planning)
  • Detection antibodies
  • Biotinylation reagent (EZ‐link Sulfo‐NHS‐LC‐Biotin, Pierce Biotechnology)
  • PBST0.5 and PBST0.1: 1× PBS containing 0.5% or 0.1% Tween‐20
  • Blocking solution:
    • PBST0.5 with 1% bovine serum albumin (BSA, for nitrocellulose‐ or amine‐coated slides)
    • 25 mM ethanolamine in sodium borate buffer, pH 9.0 (for slides with amine‐reactive groups, e.g., epoxy or NHS)
  • PBST0.5 with 1% BSA
  • PBST0.1 with 0.1% BSA
  • Streptavidin‐β‐phycoerythrin (Invitrogen) or dye‐labeled anti‐biotin (Jackson ImmunoResearch)
  • Orbital shaker, 4°C
  • Swinging‐bucket clinical centrifuge
  • Slide racks and staining chambers
  • Humidified chamber (e.g., slide box with damp paper towels)
  • Slide scanner

Basic Protocol 2: Detecting Glycans on Proteins Captured by Antibody Arrays

  Materials
  • Streptavidin‐phycoerythrin (streptavidin‐PE, Invitrogen)
  • Fluorescent antibody‐labeling reagent (e.g., Cy3‐NHS ester, GE Healthcare)
  • Additional reagents and equipment for sample capture on antibody arrays (see protocol 1)
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Figures

Videos

Literature Cited

Literature Cited
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