Protein Microarrays for Disease Analysis: Methods and Protocols (Methods in Molecular Biology, 2344)

✍ Scribed by Rodrigo Barderas (editor), Joshua LaBaer (editor), Sanjeeva Srivastava (editor)

• Publisher: Humana
• Year: 2021
• Tongue: English
• Leaves: 282
• Category: Library

✦ Synopsis

This volume focuses on protein analysis, and covers a wide array of uses of protein microarray for disease analysis. The chapters in this book discuss different stages of protein microarrays from their construction to their use, including different types of protein microarrays such as recombinant proteins, antibody, phage, and NAPPA protein microarrays, in planar format or in solution via beads arrays. Written in the highly successful Methods in Molecular Biology series format, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Comprehensive and cutting-edge, Protein Microarrays for Disease Analysis: Methods and Protocols is a valuable resource for graduate and post-doctoral fellows interested in protein microarrays, as well as senior researchers interested in gaining more insight into this developing field.

✦ Table of Contents

Preface
Contents
Editors and Contributors
Part I: Introduction
Chapter 1: Protein Microarray-Based Proteomics for Disease Analysis
1 Introduction
References
Part II: Design and Production of Protein Microarrays
Chapter 2: Systematic and Rational Design of Protein Arrays in Noncontact Printers: Pipeline and Critical Aspects
1 Introduction
2 Materials
2.1 Source Plate Preparation
2.2 Chemical Functionalization of Microarray Silica Surface
2.3 Noncontact Printing
3 Methods
3.1 Design of Customized Array Layout
3.2 Array Map Generation and Array Content Preparation
3.2.1 Originate the Well Plate Map
3.2.2 Source Plate Preparation
3.3 Chemical Functionalization of Microarray Glass Surface
3.4 Noncontact Printing
3.5 Generate the File for Analysis
3.6 Quality Control
3.7 Analysis of Results
4 Results
5 Notes
References
Chapter 3: Phage Microarrays for Screening of Humoral Immune Responses
1 Introduction
2 Materials
2.1 Common Reagents and Equipment
2.2 Phage Display
2.3 Phage Microarrays
3 Methods
3.1 Phage Display
3.1.1 Patient and Control Serum IgG Isolation
3.1.2 Amplification of T7 Phage Display Libraries
3.1.3 Removal of Phages Displaying Unspecific Peptides or Proteins (Negative Selection)
3.1.4 Enrichment of the T7 Phage Library on Specific Phages (Positive Selection)
3.1.5 Monoclonal Phage Amplification
3.2 Phage Microarrays
3.2.1 Printing
3.2.2 Hybridization
3.3 Scanning and Data Analysis
3.4 Validation
4 Notes
References
Chapter 4: Identification of Antibody Biomarker Using High-Density Nucleic Acid Programmable Protein Array
1 Introduction
2 Materials
2.1 HD-NAPPA Protein Array Expression Materials
2.2 HD-NAPPA Protein Array Expression Setup
2.3 Primary Antibodies (Serum/Plasma) Probing
2.4 Secondary Antibody Visualization and Image Scanning
3 Methods
3.1 HD-NAPPA Protein Array Expression
3.1.1 Materials and Preparation
3.1.2 Fill-n-Seal Apparatus Assembling and HD-NAPPA Expression
3.1.3 Fill-n-Seal Apparatus De-assembling
3.2 Primary (Antibody and Serum) Probing
3.3 Secondary Antibody Probing and Image Scanning
3.4 Data Analysis
3.4.1 Expression Quality Control Analysis (Fig. 3a)
3.4.2 Serology Result Analysis
4 Notes
References
Chapter 5: Bead-Based Assays for Validating Proteomic Profiles in Body Fluids
1 Introduction
2 Materials
2.1 Bead Coupling
2.2 Selection of Antibody Pairs and Assay Validation
3 Methods
3.1 Bead Coupling
3.2 Selection of Antibody Pairs
3.3 Immunoassay Optimization and Validation
4 Notes
References
Part III: Applications of Protein Microarrays
Chapter 6: Analysis of Protein-Protein Interactions by Protein Microarrays
1 Introduction
2 Materials
2.1 Common Reagents
2.2 Equipment
2.3 Protein Microarrays
2.4 Proteins
2.4.1 Protein Extraction
2.4.2 Biotin Labeling
2.4.3 Fluorophore Labeling
2.5 Detection
3 Methods
3.1 Protein Extraction and Quantification
3.2 Labeling of Protein Samples
3.2.1 Biotin Labeling
3.2.2 Fluorophore Labeling
3.3 Hybridization
3.4 Scanning and Data Analysis
3.5 Validation
4 Notes
References
Chapter 7: Detection of Posttranslational Modification Autoantibodies Using Peptide Microarray
1 Introduction
2 Materials
3 Methods
3.1 Detection of Serum Autoantibody Using Peptide Microarray
3.2 Data Analysis
3.3 Results
4 Notes
References
Chapter 8: Epitope Mapping of Allergenic Lipid Transfer Proteins
1 Introduction
2 Materials
2.1 Microarray Printing
2.2 Microarray Hybridization
2.3 Data Acquisition and Analysis
3 Methods
3.1 Ethanol Precipitation of DNA
3.2 Peptide Microarray Printing
3.3 Microarray Hybridization
3.3.1 Detection of DNA Printed on NAPPA Slides
3.3.2 Express Protein on DNA-Printed NAPPA Slides
3.3.3 Serum, PicoGreen, and Anti-HaloTag Antibody Incubation
3.3.4 IgE and IgG Detection
3.4 Scanning and Data Analysis
4 Notes
References
Chapter 9: Epitope Mapping of Food Allergens Using Noncontact Piezoelectric Microarray Printer
1 Introduction
2 Materials
2.1 Microarray Printing
2.2 Microarray Hybridization
2.3 Data Acquisition and Analysis
3 Methods
3.1 Peptide Microarray Printing
3.2 Microarray Hybridization
3.2.1 Blocking
3.2.2 Serum Incubation
3.2.3 IgE and IgG4 Detection
3.3 Data Acquisition and Analysis
3.3.1 Microarray Scanning
3.3.2 Data Analysis and Visualization
4 Notes
References
Part IV: Deciphering Immune Responses in Diseases
Chapter 10: Protein Arrays for the Identification of Seroreactive Protein Markers for Infectious Diseases
1 Introduction
2 Materials
2.1 Reagents
2.2 Equipment
2.3 Software
3 Methods
3.1 Blocking
3.2 Probing
3.3 Assay
3.4 Washing and Drying
3.5 Scanning
3.6 Data Analysis
4 Notes
References
Chapter 11: Glass Slide-Printed Protein Arrays as a Platform to Discover Serodiagnostic Antigens Against Bacterial Infections
1 Introduction
2 Materials
3 Methods
4 Notes
References
Chapter 12: Affinity Proteomics Assays for Cardiovascular and Atherosclerotic Disease Biomarkers
1 Introduction
2 Materials
2.1 General Material and Equipment
2.2 Sample Labeling Reagents
2.3 Antibody-Bead Coupling Reagents
2.4 Labeling and Coupling Efficiency Testing Reagents
2.5 Single-Binder Assay Reagents
2.6 Antibody Labeling on Beads
3 Methods
3.1 Sample Labeling Protocol
3.2 Antibody-Bead Coupling Protocol
3.3 Labeling and Coupling Efficiency Assessment
3.3.1 Antibody Coupling Efficiency
3.3.2 Sample Labeling Efficiency
3.4 Single-Binder Assay Protocol
3.5 Bead-Assisted Antibody Labeling (Detection Antibody Labeling)
4 Notes
References
Chapter 13: Nucleic Acid Programmable Protein Arrays (NAPPA) for the Discovery of Autoantibodies in Osteoarthritis
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Serum Autoantibody Profiling on NAPPA
2.3 Serum Autoantibody Profiling on ELISA
2.4 Data Analysis
3 Methods
3.1 Protein Display and Serum Autoantibody Profiling on NAPPA
3.2 Coating Plates for ELISA
3.3 Protein Expression for ELISA
3.4 Serum Autoantibody Profiling on ELISA
4 Notes
References
Chapter 14: Profiling Autoantibody Responses to Devise Novel Diagnostic and Prognostic Markers Using High-Density Protein Micr...
1 Introduction
2 Materials
2.1 Reagents
2.2 Instruments and Software
2.3 Materials
3 Methods
3.1 Recombinant Protein Production and Purification
3.2 Printing Protocol
3.3 Assay Protocol
3.4 Data Collection
3.5 Analysis
4 Concluding Remarks
5 Notes
References
Part V: Antibody Microarrays for the Characterization of Disease-Specific Protein Profiles
Chapter 15: Deciphering Intracellular Signaling Pathways in Tumoral Pathologies
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Sample Preparation for Protein Microarrays
2.2.1 Cell Lysis and Protein Extraction
2.2.2 IgG Depletion and Protein Quantification
2.2.3 Dye Labeling
2.3 Preparation of Antibody Microarrays
2.3.1 Blocking the Microarray Surface
2.3.2 Wash Microarray
2.3.3 Microarray System Incubation
2.4 Incubation of Dye-Conjugated Cell Lysates on Antibody Microarrays
2.4.1 Preparation of Sample Dilution
2.4.2 Incubation in Microarray
2.4.3 Wash and Drying Microarray
2.5 Scan of Microarrays and Data Analysis
2.6 Self-Assembled (NAPPA-ELISA) Protein Arrays
2.6.1 cDNA Preparation
2.6.2 In Situ Protein Expression
2.6.3 ELISA Test
3 Methods
3.1 Cell Culture
3.1.1 Cell Culture Conditions
3.2 Preparation of Samples
3.2.1 Extraction of Protein
3.2.2 IgG Depletion, Quantification
3.2.3 Dye Labeling
3.3 Antibody Microarray Blocking
3.3.1 Blocking the Microarray Surface
3.3.2 Wash Microarray
3.3.3 Microarray System Incubation
3.4 Incubation of Dye-Labeled Protein Lysates in Antibody Microarrays
3.4.1 Microarray Incubation
3.4.2 Washing and Drying Microarray
3.5 Microarray Scanning and Data Analysis
3.6 Self-Assembled (NAPPA-ELISA) Microtiter Protein Arrays
3.6.1 cDNA Preparation
3.6.2 In Situ Protein Expression
96-Well Plate Activation for NAPPA-ELISA
In Situ Protein Expression (IVTT)
Incubation with Dye-Labeled Protein Lysates
3.6.3 ELISA Assay of IVTT Protein
4 Results
5 Notes
References
Chapter 16: Combination of Antibody Arrays to Functionally Characterize Dark Proteins in Human Olfactory Neuroepithelial Cells
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Cell Culture
2.1.2 Cytokine and Phosphokinase Arrays
2.2 Equipment
3 Methods
3.1 Harvest, Isolation, Culture, and Maintenance of Human Olfactory Neuroepithelial Cells
3.2 Treatment with Recombinant PITHD1
3.3 Protein Extraction
3.4 Sample Incubation on Cytokine/Phosphokinase Antibody Membranes (See Note 7)
3.5 Cytokines and Phosphokinase Detection (See Note 15)
3.6 Cytokines and Phosphokinase Quantification (ImageLab Software)
3.7 Statistical Analysis of the Differential Proteins
3.8 Data Statistics (Perseus Software)
4 Notes
References
Chapter 17: Protein Microarrays for Ocular Diseases
1 Introduction
2 Main Types and Characteristics of Protein Arrays Used for Ocular Disease Research
3 Antibody Microarrays
3.1 Antibody Microarray Approaches in Ocular Surface Diseases: Dry Eye
3.2 Antibody Microarray Application in Intraocular Diseases: Age-Related Macular Degeneration
3.3 Antibody Microarray Technology in Intraocular Pathologies: Diabetic Retinopathy
3.4 Antibody Microarray Approaches in Intraocular Diseases: Glaucoma
4 Reverse-Phase Protein Arrays
4.1 Reverse-Phase Protein Microarray Technology in Age-Related Macular Degeneration
5 Protein Microarrays
5.1 Protein Microarray Technology in Age-Related Macular Degeneration and Glaucoma
6 Advantages and Drawbacks of Protein Microarray Applications to Ocular Diseases
7 Conclusions
References
Part VI: Statistical Methods for Analyzing Protein Microarray
Chapter 18: Statistical Methods for Analysis of Protein Microarray Data Using R
1 Introduction
2 Materials
3 Methods
3.1 Exploratory Data Analysis
3.2 Differential Analysis of a Single Protein
3.3 Differential Analysis of Multiple Proteins
3.4 Analysis of Protein Biomarkers
3.5 Protein Correlation Analysis
3.6 Power Analysis
References
Index