Protein Microarray for Disease Analysis: Methods and Protocols (Methods in Molecular Biology, 723)

✍ Scribed by Catherine J. Wu (editor)

• Publisher: Humana
• Year: 2011
• Tongue: English
• Leaves: 371
• Category: Library

✦ Synopsis

Protein microarrays have been used for a wide variety of important tasks, such as identifying protein-protein interactions, discovering disease biomarkers, identifying DNA-binding specificity by protein variants, and for characterization of the humoral immune response.  In Protein Microarray for Disease Analysis: Methods and Protocols, expert researchers provide concise descriptions of the methodologies currently used to fabricate microarrays for the comprehensive analysis of proteins or responses to proteins that can be used to dissect human disease.  These methodologies are the toolbox for revolutionizing drug development and cell-level biochemical understanding of human disease processes.  Beginning with a section on protein-detecting analytical microarrays, the volume continues with sections covering antigen microarrays for immunoprofiling, protein function microarrays, the validation of candidate targets, proteomic libraries, as well as signal detection strategies and data analysis techniques.  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 key tips on troubleshooting and avoiding known pitfalls.

 

Practical and cutting-edge,  Protein Microarray for Disease Analysis: Methods and Protocols serves as a solid framework to aid scientists in understanding how protein microarray technology is presently developing and how it can be applied to transform our analysis of human disease.

✦ Table of Contents

Protein Microarray for Disease Analysis
Preface
Acknowledgments
Contents
Contributors
Part I:Protein-Detecting Analytical Microarrays
Chapter 1: Detecting and Quantifying Multiple Proteins in Clinical Samples in High-Throughput Using Antibody Microarrays
1. Introduction
2. Materials
2.1. Buffers (see Note 1)
2.2. Additional Equipment and Reagents
3. Methods
3.1. Printing and Storage of the Microarray Plates
3.2. Preparation of the Mixing Plate
3.3. Preparation of the Microarray Plate
3.4. Scanning, Image Analysis, and Data Analysis
4. Notes
References
Chapter 2: Analysis of Serum Protein Glycosylation with Antibody–Lectin Microarray for High-Throughput Biomarker Screening
1. Introduction
2. Materials
2.1. Antibody–Lectin Microarray with Fluorescence Detection
2.1.1. Printing
2.1.2. Antibody Blocking
2.1.3. Hybridization of Slides
2.1.4. Slide Scanning
2.1.5. On-Slide Digestion
2.1.6. MALDI-QIT-TOF
3. Methods
3.1. Antibody Array Printing
3.2. Antibody–Lectin Array with Fluorescence Detection
3.2.1. Antibody Array Blocking
3.2.2. Optimizing Conditions
3.2.3. Experimental Design
3.2.4. Hybridization of Slides
3.2.5. Slide Scanning
3.2.6. Data Analysis
3.3. On-Slide Digestion and MALDI Sample Preparation
3.4. MALDI-MS
4. Notes
References
Chapter 3: Antibody Suspension Bead Arrays
1. Introduction
2. Materials
2.1. Bead Coupling
2.2. Sample Labeling
2.3. Assay Procedure
3. Methods
3.1. Bead Coupling
3.2. Bead Mixture Preparation
3.3. Sample Labeling
3.4. Assay Procedure
4. Notes
References
Chapter 4: Reverse Protein Arrays Applied to Host–Pathogen Interaction Studies
1. Introduction
2. Materials
2.1. Preparation of Lysates
2.1.1. In Vitro Infection
2.1.2. Cell Lysis
2.2. Sample Preparation for Spotting
2.2.1. Determination of Protein Concentration in Lysates
2.2.2. Preparation of Spotting Microplate
2.3. Microarray Spotting
2.4. Chip Blocking
2.5. Reverse Array Assay
2.6. Readout and Data Analysis
3. Methods
3.1. Preparation of Lysates
3.1.1. In Vitro Infection (see Note 4)
3.1.2. Cell Lysis (see Note 8)
3.2. Sample Preparation for Spotting
3.2.1. Determination of Protein Concentration in Lysates
3.2.2. Preparation of Reference Spotting Solution
3.2.3. Preparation of Spotting Microplate
3.3. Microarray Spotting
3.4. Chip Blocking
3.5. Reverse Protein Array Assay
3.6. Readout and Data Analysis
3.7. Data Interpretation and Quality Control
3.8. Antibody Validation
4. Notes
References
Chapter 5: Identification and Optimization of DNA Aptamer Binding Regions Using DNA Microarrays
1. Introduction
2. Materials
2.1. Aptamer Microarray
2.2. Fluorescent Labeling of IgE
2.3. Array Blocking, Probing, and Washing
3. Methods
3.1. Aptamer Microarray Design
3.2. Labeling of IgE
3.3. Array Blocking/Probing/Washing
3.4. Array Scanning and Data Analysis (Depends on Array Manufacturer)
4. Notes
References
Chapter 6: Recombinant Lectin Microarrays for Glycomic Analysis
1. Introduction
2. Materials
2.1. Cloning and Purification of Recombinant DNA
2.2. Protein Expression and Purification
2.3. ELISA Activity Assay
2.4. Recombinant Lectin Microarray
3. Methods
3.1. Cloning and Purification of Recombinant DNA
3.2. Protein Expression and Purification
3.3. ELISA Activity Assay
3.4. Recombinant Lectin Microarray
4. Notes
References
Part II:Antigen Microarrays for Immunoprofiling
Chapter 7: Recombinant Antigen Microarrays for Serum/Plasma Antibody Detection
1. Introduction
1.1. Considerations in Expression of Recombinant Antigens for Protein Microarrays
1.2. Considerations in Printing of Recombinant Antigens in Custom Protein Microarrays
1.3. Allogeneic Antibodies Against H-Y Antigens Develop After Sex-Mismatched Transplantation
1.4. H-Y Antigen Microarray Development
2. Materials
2.1. Preparation of Sera/Plasma
2.2. Recombinant Antigens for Printing
2.3. Bacterial Cell Culture and Purification of Recombinant Protein Expression
2.4. Printing the Purified Recombinant Proteins to Obtain Protein Microarrays
2.5. Probing and Developing H-Y Recombinant Protein Microarrays
2.6. Data Analysis
3. Methods
3.1. Preparation of Plasma Samples
3.2. Recombinant Antigen Design
3.3. Bacterial Cell Culture and Purification of Recombinant Protein Expression
3.4. Printing the Purified Recombinant Proteins to Obtain Protein Microarrays
3.5. Probing and Developing H-Y Recombinant Protein Microarrays
3.5.1. Probing with Anti-V5 to Detect Printed Protein Spots
3.5.2. Probing with Human Serum/Plasma
3.6. Data Analysis
4. Notes
References
Chapter 8: SPOT Synthesis as a Tool to Study Protein–Protein Interactions
1. Introduction
2. Materials
2.1. Preparation of Cellulose Membranes and SPOT Synthesis of Macroarrays
2.2. Peptide Modifications for the Preparation of Microarrays and Cleavage of Peptides from the Cellulose Membrane
2.3. Preparation of Microarrays
2.4. Detection Methods
2.4.1. Detection of Bound HRP-Labeled Protein Using Chemiluminescence
2.4.2. Detection of Bound HRP-Labeled Protein Using Staining
2.4.3. Detection of Bound AP-Labeled Protein Using Staining
2.4.4. Regeneration (Stripping) of Used Cellulose Membrane
2.4.5. Fluorescence Detection of Bound Ligands on Peptide Microarrays
3. Methods
3.1. Preparation of Cellulose Membranes and SPOT Synthesis of Macroarrays
3.1.1. Preparation of Esterified Membranes
3.1.2. Preparation of Amino-Alkyl Ether-Linked Membranes
3.1.3. Preparation of Coupling Solutions
3.1.4. SPOT Synthesis of the Peptide Array
3.2. Peptide Modifications for Preparation of Microarrays and Cleavage of Peptides from the Cellulose Membrane
3.2.1. Introduction of a Spacer
3.2.2. Coupling of a Linker Group
3.2.3. Cleavage of the Peptides from a Membrane as Free Peptide Amides
3.3. Preparation of Microarrays
3.4. Detection Methods
3.4.1. Detection of Bound HRP-Labeled Protein Using Chemiluminescence
3.4.2. Detection of Bound HRP-Labeled Protein Using Staining
3.4.3. Detection of Bound AP-Labeled Protein Using Staining
3.4.4. Regeneration (Stripping)
3.4.5. Fluorescence Detection on Peptide Microarrays
4. Notes
References
Chapter 9: Native Antigen Fractionation Protein Microarrays for Biomarker Discovery
1. Introduction
2. Materials
2.1. Construction of Fractionated Native Antigen Lysate Microarrays
2.1.1. Cell Culture and Lysis
2.1.2. Protein Fractionation
2.1.3. Microarray Printing
2.2. Sample Preparation and Microarray Experiments
2.2.1. Purification of IgG
2.2.2. Labeling of IgG with Fluorescent Dyes
2.2.3. Removal of Unbound Dye
2.2.4. Microarray Incubation with Patient IgG
2.3. Results and Analysis
2.3.1. Microarray Scanning and Quantitation
2.3.2. Statistical Analysis
3. Methods
3.1. Construction of Fractionated Native Antigen Lysate Microarrays
3.1.1. Cell Culture and Lysis
3.1.2. Protein Fractionation: First Dimension – Chromatofocusing
3.1.3. Protein Fractionation: Second Dimension – Reversed Phase HPLC
3.1.4. Microarray Printing and Blocking
3.2. Sample Preparation and Microarray Experiments
3.2.1. Purification of IgG Using Melon™ Gel Kit
3.2.2. Labeling of IgG with Fluorescent Dyes
3.2.3. Removal of Unbound Dye with Desalting Columns
3.2.4. Antibody Array Incubation with Patient IgG
3.3. Results and Analysis
3.3.1. Microarray Scanning and Quantitation (see Note 15)
3.3.2. Biostatistical Data Analysis
4. Notes
References
Chapter 10: Immunoprofiling Using NAPPA Protein Microarrays
1. Introduction
1.1. Study Design Considerations
1.1.1. Serum Samples
1.1.2. Experimental Setup
1.2. Protein Microarray Considerations
1.2.1. Spot Replicates
1.2.2. Controls
1.2.3. Technical Reproducibility Test
2. Materials
2.1. Activation of cDNA-Based Microarrays
2.2. Detection of Protein Display on the Microarrays
2.3. Serum Antibody Profiling
3. Methods
3.1. Activation of NAPPA Microarrays
3.2. Detection of Protein Display on the Microarrays
3.3. Serum Antibody Profiling
4. Notes
References
Part III:Protein Function Microarrays
Chapter 11:
High-Throughput Mammalian Two-Hybrid Screening
for Protein–Protein Interactions Using Transfected
Cell Arrays (CAPPIA)
1. Introduction
2. Materials
2.1. Expression Plasmids
2.2. Sample Preparation
2.3. Slides
2.4. Printing of the Arrays
2.5. Reverse Transfection and Fixation
2.6. Fluorescence Detection and Analysis
3. Methods
3.1. Plasmid Constructs
3.1.1. Bait and Prey Constructs
3.1.2. Reporter Constructs
3.1.3. Constructs Used to Monitor Transfection Efficiency and Array Outline
3.1.4. Preparation of Plasmid Solutions
3.2. Sample Preparation
3.3. Slide Surface
3.4. Printing of the Arrays
3.5. Reverse Transfection
3.6. Fixation
3.7. Detection and Analysis
4. Notes
References
Chapter 12: Protein–Protein Interactions: An Application of Tus-Ter Mediated Protein Microarray System
1. Introduction
2. Materials
2.1. Cloning
2.2. Expression Check
2.3. Microarray Fabrication
2.4. Quality Control of Microarray Printing
2.5. In Situ Expression of Proteins
2.6. Prehybridization and Hybridization
2.6.1. Labeling the Secondary Antibody
2.7. Detection of Expression/Interaction
3. Methods
3.1. Cloning
3.1.1. Construction of Base Microarray Plasmid
3.1.2. Construction of Target Plasmids
3.1.3. Construction of Query Plasmids
3.2. Expression Check
3.3. Microarray Fabrication
3.4. Microarray Printing: Quality Control
3.5. In Situ Expression of Protein
3.6. Prehybridization and Hybridization
3.6.1. Secondary Antibody Labeling with Cy3 and Cy5 Dyes
3.6.2. Removal of Unbound Dye
3.6.3. Secondary Antibody Hybridization
3.7. Scanning
4. Notes
References
Chapter 13: Kinase Substrate Interactions
1. Introduction
2. Materials
3. Methods
3.1. Preparing the Kinase
3.2. Blocking
3.3. Probing
3.4. Drying and Scanning the Microarray
3.5. Data Acquisition and Analysis
4. Notes
References
Chapter 14: A Functional Protein Microarray Approach to Characterizing Posttranslational Modifications on Lysine Residues
1. Introduction
2. Materials
2.1. Equipment
2.2. Protein Microarray Printing
2.3. Ubiquitylation Reaction
2.4. Protein Acetylation Reaction
3. Methods
3.1. Printing Protein Microarrays
3.2. Ubiquitylation Reaction on a Protein Microarray
3.3. Acetylation Assay on a Chip
4. Notes
References
Part IV:Strategies for Validation of Candidate Targets
Chapter 15: Multiplexed Detection of Antibodies Using Programmable Bead Arrays
1. Introduction
2. Materials
2.1. In Vitro Protein Expression
2.2. RAPID ELISA (96-Well Format)
2.3. Bead-Based Multiplexed ELISA
3. Methods
3.1. In Vitro Transcription/Translation of Recombinant Proteins
3.1.1. DNA Preparation
3.1.2. DNA Precipitation (If Not Already Done)
3.1.3. In Vitro Transcription/Translation
3.2. RAPID ELISA (96-Well Format)
3.3. Bead-Array ELISA
3.3.1. Preparation of Antibodies for Bead Conjugation
3.3.2. Antibody Coupling to Luminex Microspheres
3.3.3. Confirmation of Antibody Coupling to Microspheres
3.3.4. Bead-Array ELISA, Day 1
3.3.5. Bead-Array ELISA, Day 2
4. Notes
References
Chapter 16: A Coprecipitation-Based Validation Methodology for Interactions Identified Using Protein Microarrays
1. Introduction
2. Materials
2.1. Protein Synthesis
2.2. ImmunoŁprecipitation
2.3. SDS-Polyacrylamide Gel Electrophoresis
2.4. Western Blot
3. Methods
3.1. Microisolation of IgG from Human Sera (Optional)
3.2. Protein Synthesis for Immunoprecipitation
3.3. ImmunoŁprecipitation
3.4. SDS-PAGE Gel Separation
3.5. Western Blot
3.6. Estimating Immunoprecipitated Protein Using ImageJ (Optional)
4. Notes
References
Part V:Generation of Proteomic Libraries
Chapter 17: Development of Expression-Ready Constructs for Generation of Proteomic Libraries
1. Introduction
2. Materials
2.1. Cell Culture and Dilution
2.2. Primer Design
2.3. PCR
2.4. Agarose Gel Electrophoresis
2.5. Enzyme Digestion to Remove Vector
2.6. Purification of PCR Products using 96-Well Gel Filtration Plate Chromatography
2.7. In-Fusion Cloning Reaction
2.8. Transformation
2.9. Clone Selection and Growth
2.10. DNA Preparation by Direct Heat Lysis of Bacterial Cells
2.11. Clone Verification by Full Insert Sequencing
2.12. Informatics Processing
3. Methods
3.1. Cell Culture and Dilution (see Note 4)
3.2. Primer Design
3.3. PCR (see Note 5)
3.4. Agarose Gel Electrophoresis
3.5. Enzyme Digestion to Remove Parental Plasmid Template (for Ampicillin-Resistant Clones Only)
3.6. Purification of PCR Products Using 96-Well Gel Filtration Plate Chromatography
3.7. In-Fusion Cloning Reaction
3.8. Transformation
3.9. Clone Selection and Growth
3.10. DNA Preparation by Direct Heat Lysis of Bacterial Cells
3.11. Clone Verification by Full Insert Sequencing
3.12. Informatics Processing
4. Notes
References
Part VI:Detection Methods
Chapter 18: Reverse Phase Protein Microarrays: Fluorometric and Colorimetric Detection
1. Introduction
2. Materials
2.1. Immunostaining RPMA
2.1.1. Fluorometric Signal Detection
2.1.2. Colorimetric Signal Detection
2.2. Sypro Ruby Total Protein Assay for RPMA
2.3. Image Acquisition and Microarray Analysis
2.3.1. Fluorometric System
2.3.2. Colorimetric System
3. Methods
3.1. Immunostaining Reverse Phase Protein Microarray
3.2. Total Protein Staining: Sypro Ruby Protein Blot Stain
3.3. Image Acquisition Procedures
3.3.1. Fluorometric Image Acquisition
3.3.2. Colorimetric Image Acquisition
3.4. Microarray Data Analysis
3.4.1. Data Analysisof RPMAs withMicrovigene Software
3.5. Data Analysisof RPMAs Stainedwith DAB Using ImageQuant Software
4. Notes
References
Chapter 19: Förster Resonance Energy Transfer Methods for Quantification of Protein–Protein Interactions on Microarrays
1. The Basis of FRET Detection
2. FRET in Biomolecular Assays
3. FRET Assays on Microarrays
4. Use of Time-Resolved FRET to Detect Protein Interactions
5. Concluding Remarks and Outlook
References
Chapter 20: Label-Free Detection with Surface Plasmon Resonance Imaging
1. Introduction
2. Materials
2.1. Reagents
2.2. Equipment
2.3. Biological Materials
3. Methods
3.1. Preparation of Secretome Samples
3.2. Printing Antibody Arrays
3.3. Binding Experiment
3.4. Data Analysis
4. Notes
References
Part VII:Data Analysis Techniques for Protein Function Microarrays
Chapter 21: Data Processing and Analysis for Protein Microarrays
1. Introduction
2. Materials
2.1. Analytical Tools
2.1.1. Determinationof Spot Intensity
2.1.2. Z-Score Analysis
2.1.3. Concentration-Dependent Analysis
2.1.4. Differential Expression Analysis
2.1.5. Differential Expression Analysis with Repeat Experiments
2.2. Software Tools
2.2.1. GenePix Pro
2.2.2. Prospector
2.2.3. ProtMAT
2.2.4. Significance Analysis of Microarrays
3. Methods
3.1. Signal Acquisition
3.2. Format Preparation
3.3. Hit Calling
3.4. Comparing Multiple Microarrays
4. Notes
References
Chapter 22: Database Resources for Proteomics-Based Analysis of Cancer
1. Introduction
2. Databases
2.1. Gene/Protein Expression Databases
2.2. Gene Mutation and SNP Databases
2.2.1. Databases that Catalog Tumor-Associated Mutations
2.2.2. SNP Databases
2.2.3. Databases Cataloguing other Genetic Abnormalities
2.3. Tumor Antigen Databases
2.4. Databases of Cancer-Associated Genes
2.5. Protein Interaction and Pathway Databases
3. Discussion
References
Index