Glycan Microarrays: Methods and Protocols (Methods in Molecular Biology, 2460)

✍ Scribed by Michelle Kilcoyne (editor), Jared Q. Gerlach (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 254
• Category: Library

✦ Synopsis

This volume encompasses short targeted reviews and methods featuring the construction and use of glycan microarray platforms. Chapters guider readers through glycan microarrays comprised of synthetic glycans, natural biomolecules, bacteria, neoglycoproteins, various applications and progress of these platforms. Written in the format of the highly successful Methods in Molecular Biology series, each chapter includes an introduction to the topic, lists necessary materials and reagents, includes tips on troubleshooting and known pitfalls, and step-by-step, readily reproducible protocols.

Authoritative and cutting-edge, Glycan Microarrays: Methods and Protocols aims to highlight the interdisciplinary nature of the development and use of the glycan microarray platform.

✦ Table of Contents

Preface
Contents
Contributors
Part I: Glycan Microarray Platforms
Chapter 1: Neoglycoprotein and Glycoprotein Printing on a Hydrogel Functionalized Microarray Surface and Incubation with Label...
1 Introduction
2 Materials
2.1 Printing Neoglycoprotein and Glycoprotein Microarray Slides
2.2 Incubation of Labeled Lectins on Neoglycoprotein and Glycoprotein Microarray Slide
3 Methods
3.1 Printing Neoglycoprotein and Glycoprotein Microarray Slides
3.1.1 Starting up Microarray Printer
3.1.2 Printing Neoglycoprotein and Glycoprotein Microarray Slides
3.2 Incubation of Labeled Lectins on Neoglycoprotein and Glycoprotein Microarray Slide
3.3 Washing Gasket Slides for Re-use
4 Notes
References
Chapter 2: Evaluation of Glycan-Binding Specificity by Glycoconjugate Microarray with an Evanescent-Field Fluorescence Detecti...
1 Introduction
2 Materials
3 Methods
3.1 Direct Labeling Method (Fig. 1) (See Note 1)
3.2 Antibody Precomplex Method (Fig. 1) (See Note 2)
3.3 Antibody Overlay Method (Fig. 1) (See Note 3)
4 Notes
References
Chapter 3: Multiplex Glycan Bead Array (MGBA) for High Throughput and High Content Analyses of Glycan-Binding Proteins Includi...
1 Introduction
2 Materials
2.1 Buffers
2.2 Blocking Buffers
2.3 Carboxylated Microspheres and Instrument
2.4 Conjugation Reagents
2.5 Lectins, Anti-Glycan Antibodies, and Glycan-Binding Proteins
2.6 Free Amine Group Containing Glycans
3 Methods
3.1 Conjugation of Glycans to Microbeads
3.2 Characterization of Glycan-Binding Epitopes Using Lectins
3.3 Analyses of Monoclonal and Polyclonal Anti-Glycan Antibodies
3.4 Glycan-Binding Protein (GBP) Assay
3.5 Profiling of Natural Anti-Glycan IgM in Human Serum
3.6 Measurement of Natural Anti-Glycan IgG Antibodies in Human Serum
References
Chapter 4: Nanocube-Based Fluidic Glycan Array
1 Introduction
2 Materials
2.1 Synthesis of Plasmonic Silver Nanocube Sensor
2.2 Synthesis of Silver-Silica Core-Shell (Ag@SiO2) Nanocube Sensor
2.3 Preparation of Lipid Bilayer Coated Ag@SiO2 Nanocubes
2.4 Ag@SiO2 Nanocube Sensor Calibration
2.5 Analyses of Lectin Binding
3 Methods
3.1 Synthesis of Plasmonic Nanocube Sensor
3.1.1 Synthesis of Nanocubes
3.1.2 Nanocube Purification
3.2 Synthesis of Silver-Silica Core-Shell (Ag@SiO2) Nanocube Sensor
3.3 Preparation of Lipid Bilayer Coated Ag@SiO2 Nanocubes
3.3.1 Preparation of Lipid Solutions
3.3.2 Preparation of SUV Solutions
Extrusion
Tip Sonication
3.3.3 Lipid Bilayer Coating on the Ag@SiO2 Nanocubes
3.4 Sensor Calibration
3.4.1 Measurement of Localized Surface Plasmon Resonance (LSPR)
3.4.2 Calibration of Sensor Sensitivity (Glycerol-Water Test)
3.4.3 Protein Binding Density Calibration (Streptavidin-Biotinyl Lipid Calibration)
3.5 Analyses of Lectin Binding
4 Notes
References
Part II: Glycan Microarray Data Extraction, Processing and use for Measurements
Chapter 5: General Strategies for Glycan Microarray Data Processing and Analysis
1 Introduction
2 Materials
2.1 Reagents and Materials
2.2 Equipment
2.3 Software
3 Methods
3.1 Creating a GAL File for a 3-Subarray Microarray Format
3.1.1 Create 3 Blocks with the Appropriate Number of Rows and Columns in GenePix
3.2 Pre-Assay Scan of the Microarray Slide
3.3 Blocking the Glycan Microarray Slide
3.4 Post-Assay Scan of the Microarray Slide
3.5 Grid Alignment and Feature Sizing
3.5.1 Coarse Alignment to the Pre-Assay Image
3.5.2 Coarse Grid Alignment to the Post-Assay Image
3.5.3 Fine Alignment and Feature Resizing of the Post-Assay Image
3.6 Identification of Problematic/Artifact Spots
3.6.1 Scratches, Lint, and Other Artifacts that Obscure Spots
3.6.2 Large Differences for Replicate Spots
3.6.3 Donuts or Ring-like Spots
3.6.4 Comet Tails
3.7 Processing and Analysis of Microarray Data
3.7.1 Extracting Data from GenePix into Excel
3.7.2 Final Processing of the Microarray Data
3.7.3 Analysis of Data Quality: Batch QC
3.7.4 Analysis of Data Quality: Experimental QC
3.8 Normalization and Scaling of Data
4 Notes
References
Chapter 6: Calculating Half Maximal Inhibitory Concentration (IC50) Values from Glycomics Microarray Data Using GraphPad Prism
1 Introduction
2 Materials
2.1 Fluorescently Labeling MAA Lectin
2.2 Binding Inhibition Incubations of MAA-AF555 on Glycan Microarray Slides
2.3 Calculation of IC50 Values from Glycomics Microarray Data
3 Methods
3.1 Fluorescently Labeling MAA Lectin
3.2 Binding Inhibition Incubations of MAA-AF555 on Glycan Microarray Slides
3.3 Calculation of IC50 Value Using Inhibition Data from Glycan Microarray Inhibition Experiments
4 Notes
References
Part III: Complex Natural and Synthetic Glycoconjugate Microarrays
Chapter 7: Synthetic Plant Glycan Microarrays as Tools for Plant Biology
Abbreviations
1 Introduction
2 Materials
3 Methods
3.1 Glycan Array Printing
3.2 Incubation of the Array with Glycan-Binding Proteins (GBPs)
4 Notes
References
Chapter 8: Mucin Purification and Printing Natural Mucin Microarrays
1 Introduction
2 Materials
2.1 Mucin Purification
2.1.1 Mucus Dissolution, Reduction, and Alkylation
2.1.2 Isopycnic Density Gradient Centrifugation in Cesium Chloride
2.1.3 Mucin Purification and Desalting by Gel Permeation Chromatography
2.2 Printing Mucin Microarray Slides
3 Methods
3.1 Mucin Purification
3.1.1 Mucus Dissolution, Reduction, and Alkylation
3.1.2 Isopycnic Density Gradient Centrifugation in Cesium Chloride
3.1.3 Mucin Purification and Desalting by Gel Permeation Chromatography
3.2 Printing Mucin Microarray Slides
4 Notes
References
Chapter 9: Bacterial Microarrays for Examining Bacterial Glycosignatures and Recognition by Host Lectins
1 Introduction
2 Materials
2.1 Fluorescent Labeling of Bacteria
2.2 Preparation of Microarrays
2.3 Microarray Binding Assays
2.4 Microarray Scanning and Data Analysis
3 Methods
3.1 Fluorescent Labeling of Bacteria
3.2 Preparation of Microarrays (See Note 12)
3.3 Microarray Binding Assays (See Note 12)
3.4 Microarray Scanning and Data Analysis
4 Notes
References
Chapter 10: Tissue Glycome Mapping: Lectin Microarray-Based Differential Glycomic Analysis of Formalin-Fixed Paraffin-Embedded...
1 Introduction
2 Materials
2.1 Preparation of FFPE Tissue Sections for Tissue Dissection
2.2 Deparaffinization and Hematoxylin Staining of FFPE Tissue Sections
2.3 LMD-Assisted Tissue Section
2.4 Tissue Protein Extraction for LMA Analysis
2.5 Fluorescence Labeling and LMA Analysis of Tissue Protein Samples
2.6 Deparaffinization and Fluorescence Histochemical Staining of FFPE Tissue Sections
3 Methods
3.1 LMD-LMA Analysis of Hematoxylin-Stained Tissue Sections
3.1.1 Preparation of Tissue Sections
3.1.2 Hematoxylin Staining
3.1.3 LMD-Assisted Tissue Dissection
3.1.4 Protein Extraction and Fluorescence Labeling
3.1.5 LMA Analysis of Cy3-Labeled Protein Samples
3.1.6 LMA Data Processing
3.2 Fluorescence Double-Staining with a Lectin and an Antibody
3.3 Fluorescence LMD-LMA Analysis of Histochemical Sections Stained with a Lectin or an Antibody
4 Notes
References
Part IV: Applications for Glycomics Microarrays
Chapter 11: Detection of Autoantibodies Using Combinatorial Glycolipid Microarrays
1 Introduction
2 Materials
2.1 Array Slide Preparation
2.2 Array Printing
2.3 Immunoassay
2.4 Scanning and Analysis
3 Method
3.1 Slide Preparation
3.2 Glycolipid Array Settings and Print Program
3.3 Immunoassay
3.4 Array Scanning and Analysis
4 Notes
References
Chapter 12: Glycan Microarrays Containing Synthetic Streptococcus pneumoniae CPS Fragments and Their Application to Vaccine De...
1 Introduction
2 Materials
2.1 Preparation of Glycan Arrays Containing Fragments of the Capsular Polysaccharide of S. pneumoniae
2.2 Serum Samples Screening Method
2.3 Immunological Evaluation of Vaccine Candidates
3 Methods
3.1 Preparation of Glycan Arrays Containing Fragments of the Capsular Polysaccharide of S. pneumoniae
3.2 Glycan Array-Based Serum Samples Screening for Evaluation of Potential Glycan Epitope
3.3 Immunological Evaluation of Vaccine Candidates
4 Notes
References
Chapter 13: Lectin-Based Protein Microarray for the Glycan Analysis of Colorectal Cancer Biomarkers: The Insulin-Like Growth F...
1 Introduction
2 Materials
2.1 Preparation of Serum and Colon Tissue Samples
2.2 Isolation of Proteins by Immunoprecipitation
2.3 Lectin-Based Protein Microarray
3 Methods
3.1 Sample Collection
3.2 Preparation of Serum and Colon Tissue Samples
3.3 Immunoprecipitation of Glycoproteins
3.4 Lectin-Based Protein Microarray
4 Notes
References
Chapter 14: Bacterial Staining and Profiling for Glycan Interactions on Glycan Microarrays for t-Test Calculation
1 Introduction
2 Materials
2.1 Fluorescently Labeling Bacteria
2.2 Bacterial Incubations on Glycan Microarray Slides
2.3 Performing Student´s t-Test in Excel Using Glycomics Microarray Data
3 Methods
3.1 Fluorescently Labeling Bacteria
3.2 Bacterial Incubations on Glycan Microarray Slides
3.3 Performing Student´s t-Test in Excel Using Glycomics Microarray Data
4 Notes
References
Chapter 15: Preparation and Fluorescent Labeling of Cell-Derived Micelles and Profiling on Glycan Microarrays
1 Introduction
2 Materials
2.1 Cell Preparation
2.2 Cell-Derived Micelle Preparation and Labeling
2.3 Profiling Cell-Derived Micelles on Glycan Microarrays
3 Method
3.1 Cell Preparation
3.2 Cell-Derived Micelle Preparation and Labeling
3.3 Profiling Fluorescently Labeled Cell-Derived Micelles on Glycan Microarrays
4 Notes
References
Index