Current Protocols in Protein Science

Current Protocols in Protein Science

Online ISBN: 9780471140863

DOI: 10.1002/0471140864

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  1. Foreword
  2. Preface
  3. Selected Suppliers of Reagents and Equipment
  4. Chapter 1 Strategies of Protein Purification and Characterization
    1. UNIT 1.1 Overview of Protein Purification and Characterization
    2. UNIT 1.2 Strategies for Protein Purification
    3. UNIT 1.3 Protein Purification Flow Charts
    4. UNIT 1.4 Purification of Glutamate Dehydrogenase from Liver and Brain
    5. UNIT 1.5 Overview of the Physical State of Proteins within Cells
  5. Chapter 2 Computational Analysis
    1. Introduction
    2. UNIT 2.1 Computational Methods for Protein Sequence Comparison and Search
    3. UNIT 2.2 Hydrophobicity Profiles for Protein Sequence Analysis
    4. UNIT 2.3 Computational Prediction of Protein Secondary Structure from Sequence
    5. UNIT 2.4 Internet Basics
    6. UNIT 2.5 Sequence Similarity Searching Using the BLAST Family of Programs
    7. UNIT 2.6 Protein Databases on the Internet
    8. UNIT 2.7 Protein Tertiary Structure Prediction
    9. UNIT 2.8 Protein Tertiary Structure Modeling
    10. UNIT 2.9 Comparative Protein Structure Modeling Using MODELLER
    11. UNIT 2.10 Protein Charge Determination
    12. UNIT 2.11 Basic Protein Sequence Analysis
    13. UNIT 2.12 Minimotif Miner: A Computational Tool to Investigate Protein Function, Disease, and Genetic Diversity
    14. UNIT 2.13 Protein Structure Analysis Online
    15. UNIT 2.14 Protein Structural Domains: Definition and Prediction
    16. UNIT 2.15 Bioinformatics Protocols in Glycomics and Glycoproteomics
    17. UNIT 2.16 Computational Prediction of Intrinsic Disorder in Proteins
  6. Chapter 3 Detection and Assay Methods
    1. Introduction
    2. UNIT 3.1 Spectrophotometric Determination of Protein Concentration
    3. UNIT 3.2 Quantitative Amino Acid Analysis
    4. UNIT 3.3 In Vitro Radiolabeling of Peptides and Proteins
    5. UNIT 3.4 Assays for Determination of Protein Concentration
    6. UNIT 3.5 Kinetic Assay Methods
    7. UNIT 3.6 Protein Biotinylation
    8. UNIT 3.7 Metabolic Labeling with Amino Acids
    9. UNIT 3.8 Analysis of Selenocysteine-Containing Proteins
    10. UNIT 3.9 Computational Large-Scale Mapping of Protein-Protein Interactions Using Structural Complexes
    11. UNIT 3.10 Microvolume Spectrophotometric and Fluorometric Determination of Protein Concentration
    12. UNIT 3.11 Quantitative Analysis of Surface Expression of Membrane Proteins Using Cold-Adapted Proteases
  7. Chapter 4 Extraction, Stabilization, and Concentration
    1. Introduction
    2. UNIT 4.1 Overview of Cell Fractionation
    3. UNIT 4.2 Purification of Organelles from Mammalian Cells
    4. UNIT 4.3 Subcellular Fractionation of Tissue Culture Cells
    5. UNIT 4.4 Desalting, Concentration, and Buffer Exchange by Dialysis and Ultrafiltration
    6. UNIT 4.5 Selective Precipitation of Proteins
    7. UNIT 4.6 Long-Term Storage of Proteins
    8. UNIT 4.7 Extraction of Proteins from Plant Tissues
    9. UNIT 4.8 The Use of Detergents to Purify Membrane Proteins
    10. UNIT 4.9 Trehalose and Protein Stability
  8. Chapter 5 Production of Recombinant Proteins
    1. Introduction
    2. UNIT 5.1 Production of Recombinant Proteins in Escherichia coli
    3. UNIT 5.2 Selection of Escherichia coli Expression Systems
    4. UNIT 5.3 Fermentation and Growth of Escherichia coli for Optimal Protein Production
    5. UNIT 5.4 Overview of the Baculovirus Expression System
    6. UNIT 5.5 Protein Expression in the Baculovirus System
    7. UNIT 5.6 Overview of Protein Expression in Saccharomyces cerevisiae
    8. UNIT 5.7 Overview of Protein Expression in Pichia pastoris
    9. UNIT 5.8 Culture of Yeast for the Production of Heterologous Proteins
    10. UNIT 5.9 Overview of Protein Expression by Mammalian Cells
    11. UNIT 5.10 Production of Recombinant Proteins in Mammalian Cells
    12. UNIT 5.11 Overview of the Vaccinia Virus Expression System
    13. UNIT 5.12 Preparation of Cell Cultures and Vaccinia Virus Stocks
    14. UNIT 5.13 Generation of Recombinant Vaccinia Viruses
    15. UNIT 5.14 Characterization of Recombinant Vaccinia Viruses and Their Products
    16. UNIT 5.15 Gene Expression Using the Vaccinia Virus/ T7 RNA Polymerase Hybrid System
    17. UNIT 5.16 Choice of Cellular Protein Expression System
    18. UNIT 5.17 Use of the Gateway System for Protein Expression in Multiple Hosts
    19. UNIT 5.18 Wheat Germ Cell-Free Expression System for Protein Production
    20. UNIT 5.19 Overview on the Expression of Toxic Gene Products in Escherichia coli
    21. UNIT 5.20 MultiBac: Multigene Baculovirus-Based Eukaryotic Protein Complex Production
    22. UNIT 5.21 Recombinant Protein Complex Expression in E. coli
    23. UNIT 5.22 A Bacterial Cell-Free Expression System to Produce Membrane Proteins and Proteoliposomes: From cDNA to Functional Assay
    24. UNIT 5.23 Autoinduction of Protein Expression
    25. UNIT 5.24 Strategies to Optimize Protein Expression in E. coli
    26. UNIT 5.25 Overview of Approaches to Preventing and Avoiding Proteolysis During Expression and Purification of Proteins
    27. UNIT 5.26 Use of the SHuffle Strains in Production of Proteins
  9. Chapter 6 Purification of Recombinant Proteins
    1. Introduction
    2. UNIT 6.1 Overview of the Purification of Recombinant Proteins
    3. UNIT 6.2 Preparation of Soluble Proteins from Escherichia coli
    4. UNIT 6.3 Preparation and Extraction of Insoluble (Inclusion-Body) Proteins from Escherichia coli
    5. UNIT 6.4 Overview of Protein Folding
    6. UNIT 6.5 Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli
    7. UNIT 6.6 Expression and Purification of GST Fusion Proteins
    8. UNIT 6.7 Expression and Purification of Thioredoxin Fusion Proteins
    9. UNIT 6.8 Automated Large-Scale Purification of a Recombinant G-Protein-Coupled Neurotensin Receptor
    10. UNIT 6.9 Overview on Concepts and Applications of Fab Antibody Fragments
    11. UNIT 6.10 E. coli Expression and Purification of Fab Antibody Fragments
    12. UNIT 6.11 Elastin-Like Polypeptides as a Purification Tag for Recombinant Proteins
    13. UNIT 6.12 Removal of Detergents from Proteins and Peptides in a Spin-Column Format
    14. UNIT 6.13 Obtaining Soluble Folded Proteins from Inclusion Bodies Using Sarkosyl, Triton X-100, and CHAPS: Application to LB and M9 Minimal Media
    15. UNIT 6.14 N-Terminal Methionine Processing
  10. Chapter 7 Characterization of Recombinant Proteins
    1. Introduction
    2. UNIT 7.1 Overview of the Characterization of Recombinant Proteins
    3. UNIT 7.2 Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy
    4. UNIT 7.3 Determining the Identity and Structure of Recombinant Proteins
    5. UNIT 7.4 Transverse Urea-Gradient Gel Electrophoresis
    6. UNIT 7.5 Analytical Ultracentrifugation
    7. UNIT 7.6 Determining the CD Spectrum of a Protein
    8. UNIT 7.7 Determining the Fluorescence Spectrum of a Protein
    9. UNIT 7.8 Light Scattering
    10. UNIT 7.9 Measuring Protein Thermostability by Differential Scanning Calorimetry
    11. UNIT 7.10 Characterizing Recombinant Proteins Using HPLC Gel Filtration and Mass Spectrometry
    12. UNIT 7.11 Rapid Screening of E. coli Extracts by Heteronuclear NMR
    13. UNIT 7.12 Determination of Membrane Protein Molecular Weight Using Sedimentation Equilibrium Analytical Ultracentrifugation
    14. UNIT 7.13 Methods for the Design and Analysis of Sedimentation Velocity and Sedimentation Equilibrium Experiments with Proteins
  11. Chapter 8 Conventional Chromatographic Separations
    1. Introduction
    2. UNIT 8.1 Overview of Conventional Chromatography
    3. UNIT 8.2 Ion-Exchange Chromatography
    4. UNIT 8.3 Gel-Filtration Chromatography
    5. UNIT 8.4 Hydrophobic-Interaction Chromatography
    6. UNIT 8.5 Chromatofocusing
    7. UNIT 8.6 Hydroxylapatite Chromatography
    8. UNIT 8.7 Reversed-Phase High Performance Liquid Chromatography of Proteins
    9. UNIT 8.8 Expanded-Bed Adsorption Chromatography
    10. UNIT 8.9 Displacement Chromatography of Proteins
    11. UNIT 8.10 Isolation of Monoclonal Antibody Charge Variants by Displacement Chromatography
  12. Chapter 9 Affinity Purification
    1. Introduction
    2. UNIT 9.1 Lectin Affinity Chromatography
    3. UNIT 9.2 Dye Affinity Chromatography
    4. UNIT 9.3 Affinity Purification of Natural Ligands
    5. UNIT 9.4 Metal-Chelate Affinity Chromatography
    6. UNIT 9.5 Immunoaffinity Chromatography
    7. UNIT 9.6 Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography
    8. UNIT 9.7 Purification of DNA-Binding Proteins Using Biotin/Streptavidin Affinity Systems
    9. UNIT 9.8 Immunoprecipitation
    10. UNIT 9.9 Overview of Affinity Tags for Protein Purification
    11. UNIT 9.10 Protein Purification Using PDZ Affinity Chromatography
  13. Chapter 10 Electrophoresis
    1. Introduction
    2. UNIT 10.1 One-Dimensional SDS Gel Electrophoresis of Proteins
    3. UNIT 10.2 One-Dimensional Isoelectric Focusing of Proteins in Slab Gels
    4. UNIT 10.3 One-Dimensional Electrophoresis Using Nondenaturing Conditions
    5. UNIT 10.4 Two-Dimensional Gel Electrophoresis
    6. UNIT 10.5 Protein Detection in Gels Using Fixation
    7. UNIT 10.6 Protein Detection in Gels Without Fixation
    8. UNIT 10.7 Electroblotting from Polyacrylamide Gels
    9. UNIT 10.8 Detection of Proteins on Blot Membranes
    10. UNIT 10.9 Capillary Electrophoresis of Proteins and Peptides
    11. UNIT 10.10 Immunoblotting and Immunodetection
    12. UNIT 10.11 Autoradiography
    13. UNIT 10.12 Overview of Digital Electrophoresis Analysis
    14. UNIT 10.13 Capillary Electrophoresis of Peptides and Proteins Using Isoelectric Buffers
  14. Chapter 11 Chemical Analysis
    1. Introduction
    2. UNIT 11.1 Enzymatic Digestion of Proteins in Solution
    3. UNIT 11.2 Enzymatic Digestion of Proteins on PVDF Membranes
    4. UNIT 11.3 Enzymatic Digestion of Proteins in Gels for Mass Spectrometric Identification and Structural Analysis
    5. UNIT 11.4 Chemical Cleavage of Proteins in Solution
    6. UNIT 11.5 Chemical Cleavage of Proteins on Membranes
    7. UNIT 11.6 Separation of Peptides on HALO 2-Micron Particles
    8. UNIT 11.7 Removal of N-Terminal Blocking Groups from Proteins
    9. UNIT 11.8 C-Terminal Sequence Analysis
    10. UNIT 11.9 Amino Acid Analysis
    11. UNIT 11.10 N-Terminal Sequence Analysis of Proteins and Peptides
    12. UNIT 11.11 Determination of Disulfide-Bond Linkages in Proteins
  15. Chapter 12 Post-Translational Modification: Glycosylation
    1. Introduction
    2. UNIT 12.1 Overview of Glycoconjugate Analysis
    3. UNIT 12.2 Metabolic Radiolabeling of Animal Cell Glycoconjugates
    4. UNIT 12.3 Inhibition of N-Linked Glycosylation
    5. UNIT 12.4 Endoglycosidase and Glycoamidase Release of N-Linked Glycans
    6. UNIT 12.5 Detection of Glycophospholipid Anchors on Proteins
    7. UNIT 12.6 Determining the Structure of Oligosaccharides N- and O-Linked to Glycoproteins
    8. UNIT 12.7 Determining the Structure of Glycan Moieties by Mass Spectrometry
    9. UNIT 12.8 Detection and Analysis of Proteins Modified by O-Linked N-Acetylglucosamine
    10. UNIT 12.9 High-Throughput Lectin Microarray-Based Analysis of Live Cell Surface Glycosylation
    11. UNIT 12.10 Preparation and Analysis of Glycan Microarrays
    12. UNIT 12.11 Use of CID/ETD Mass Spectrometry to Analyze Glycopeptides
    13. UNIT 12.12 Protein O-Mannosylation in Metazoan Organisms
  16. Chapter 13 Post-Translational Modification: Phosphorylation and Phosphatases
    1. Introduction
    2. UNIT 13.1 Overview of Protein Phosphorylation
    3. UNIT 13.2 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
    4. UNIT 13.3 Phosphoamino Acid Analysis
    5. UNIT 13.4 Detection of Phosphorylation by Immunological Techniques
    6. UNIT 13.5 Detection of Phosphorylation by Enzymatic Techniques
    7. UNIT 13.6 Preparation and Application of Polyclonal and Monoclonal Sequence-Specific Anti-Phosphoamino Acid Antibodies
    8. UNIT 13.7 Assays of Protein Kinases Using Exogenous Substrates
    9. UNIT 13.8 Permeabilization Strategies to Study Protein Phosphorylation
    10. UNIT 13.9 Phosphopeptide Mapping and Identification of Phosphorylation Sites
    11. UNIT 13.10 Use of Protein Phosphatase Inhibitors
    12. UNIT 13.11 Tyrosine Phosphorylation Enrichment and Subsequent Analysis by MALDI-TOF/TOF MS/MS and LC-ESI-IT-MS/MS
    13. UNIT 13.12 In-Gel Phosphatase Assay Using Fluorogenic and Radioactive Substrates
  17. Chapter 14 Post-Translational Modification: Specialized Applications
    1. Introduction
    2. UNIT 14.1 Analysis of Disulfide Bond Formation
    3. UNIT 14.2 Analysis of Protein Acylation
    4. UNIT 14.3 Analysis of Protein Prenylation In Vitro and In Vivo Using Functionalized Phosphoisoprenoids
    5. UNIT 14.4 Analysis of Oxidative Modification of Proteins
    6. UNIT 14.5 Analysis of Protein Ubiquitination
    7. UNIT 14.6 Analysis of Protein S-Nitrosylation
    8. UNIT 14.7 Detection of Tyrosine Sulfation on Proteins
    9. UNIT 14.8 Analysis of Protein Sumoylation
    10. UNIT 14.9 Identifying and Quantifying Sites of Protein Methylation by Heavy Methyl SILAC
    11. UNIT 14.10 Analysis of Histone Modifications by Mass Spectrometry
    12. UNIT 14.11 Assays for Acetylation and Other Acylations of Lysine Residues
    13. UNIT 14.12 Determination of Protein Lysine Deacetylation
    14. UNIT 14.13 Enrichment and Detection of Tyrosine-Nitrated Proteins
    15. UNIT 14.14 Fluorescent Labeling of Specific Cysteine Residues Using CyMPL
    16. UNIT 14.15 Nonradioactive Analysis of Dynamic Protein Palmitoylation
    17. UNIT 14.16 Detection and Measurement of Methionine Oxidation in Proteins
  18. Chapter 15 Chemical Modification of Proteins
    1. Introduction
    2. UNIT 15.1 Modification of Cysteine
    3. UNIT 15.2 Modification of Amino Groups
    4. UNIT 15.3 Site-Specific Protein Labeling via Sortase-Mediated Transpeptidation
    5. UNIT 15.4 Application of meta- and para-Phenylenediamine as Enhanced Oxime Ligation Catalysts for Protein Labeling, PEGylation, Immobilization, and Release
  19. Chapter 16 Mass Spectrometry
    1. Introduction
    2. UNIT 16.1 Overview of Peptide and Protein Analysis by Mass Spectrometry
    3. UNIT 16.2 Matrix-Assisted Laser Desorption/Ionization Time-of-Flight Mass Analysis of Peptides
    4. UNIT 16.3 Sample Preparation for MALDI Mass Analysis of Peptides and Proteins
    5. UNIT 16.4 Mass Spectrometry-Based Bottom-Up Proteomics: Sample Preparation, LC-MS/MS Analysis, and Database Query Strategies
    6. UNIT 16.5 Searching Sequence Databases Over the Internet: Protein Identification Using MS-Fit
    7. UNIT 16.6 Searching Sequence Databases Over the Internet: Protein Identification Using MS-Tag
    8. UNIT 16.7 Enzymatic Approaches for Obtaining Amino Acid Sequence: On-Target Ladder Sequencing
    9. UNIT 16.8 Introducing Samples Directly into Electrospray Ionization Mass Spectrometers by Direct Infusion Using a Nanoelectrospray Interface
    10. UNIT 16.9 Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using Microscale Capillary Liquid Chromatography
    11. UNIT 16.10 Protein Identification Using a Quadrupole Ion Trap Mass Spectrometer and SEQUEST Database Matching
    12. UNIT 16.11 De Novo Peptide Sequencing via Manual Interpretation of MS/MS Spectra
    13. UNIT 16.12 Rapid Detergent Removal from Peptide Samples with Ethyl Acetate for Mass Spectrometry Analysis
    14. UNIT 16.13 Preparation and Analysis of Proteins and Peptides Using MALDI TOF/TOF Mass Spectrometry
  20. Chapter 17 Structural Biology
    1. Introduction
    2. UNIT 17.1 Overview of Protein Structural and Functional Folds
    3. UNIT 17.2 Electron Microscopy and Image Processing: Essential Tools for Structural Analysis of Macromolecules
    4. UNIT 17.3 Principles of Macromolecular X-Ray Crystallography
    5. UNIT 17.4 Crystallization of Macromolecules
    6. UNIT 17.5 Overview on the Use of NMR to Examine Protein Structure
    7. UNIT 17.6 Investigating Solution-Phase Protein Structure and Dynamics by Hydrogen Exchange Mass Spectrometry
    8. UNIT 17.7 Introduction to Atomic Force Microscopy (AFM) in Biology
    9. UNIT 17.8 Raman Spectroscopy of Proteins and Nucleoproteins
    10. UNIT 17.9 Crystallization of Integral Membrane Proteins
    11. UNIT 17.10 Application of Dynamic Light Scattering in Protein Crystallization
    12. UNIT 17.11 The STINT-NMR Method for Studying In-cell Protein-Protein Interactions
    13. UNIT 17.12 Native Mass Spectrometry as a Tool in Structural Biology
    14. UNIT 17.13 Cryo-Electron Tomography for Structural Characterization of Macromolecular Complexes
    15. UNIT 17.14 Sample Preparation, Data Collection, and Preliminary Data Analysis in Biomolecular Solution X-Ray Scattering
    16. UNIT 17.15 Overview of Electron Crystallography of Membrane Proteins: Crystallization and Screening Strategies Using Negative Stain Electron Microscopy
    17. UNIT 17.16 Neutron Scattering Techniques and Applications in Structural Biology
    18. UNIT 17.17 Pulsed EPR Distance Measurements in Soluble Proteins by Site-Directed Spin Labeling (SDSL)
    19. UNIT 17.18 Overview of Probing Protein-Ligand Interactions Using NMR
  21. Chapter 18 Preparation and Handling of Peptides
    1. Introduction
    2. UNIT 18.1 Introduction to Peptide Synthesis
    3. UNIT 18.2 Combinatorial Peptide Synthesis on a Microchip
    4. UNIT 18.3 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins
    5. UNIT 18.4 Native Chemical Ligation of Polypeptides
    6. UNIT 18.5 Synthesis and Application of Peptide Dendrimers As Protein Mimetics
    7. UNIT 18.6 Disulfide Bond Formation in Peptides
    8. UNIT 18.7 Guide for Resin and Linker Selection in Solid-Phase Peptide Synthesis
    9. UNIT 18.8 Overview of Solid Phase Synthesis of “Difficult Peptide” Sequences
    10. UNIT 18.9 Phage Display for Generating Peptide Reagents
    11. UNIT 18.10 Utilizing Peptide SPOT Arrays to Identify Protein Interactions
    12. UNIT 18.11 Expression and Purification of ZEBRA Fusion Proteins and Applications for the Delivery of Macromolecules into Mammalian Cells
    13. UNIT 18.12 Storage and Handling Guidelines for Custom Peptides
    14. UNIT 18.13 Peptidomic Approaches to Study Proteolytic Activity
  22. Chapter 19 Identification of Protein Interactions
    1. Introduction
    2. UNIT 19.1 Analysis of Protein-Protein Interactions
    3. UNIT 19.2 Interaction Trap/Two-Hybrid System to Identify Interacting Proteins
    4. UNIT 19.3 Phage-Based Expression Cloning to Identify Interacting Proteins
    5. UNIT 19.4 Detection of Protein-Protein Interactions by Coprecipitation
    6. UNIT 19.5 Imaging Protein-Protein Interactions by Förster Resonance Energy Transfer (FRET) Microscopy in Live Cells
    7. UNIT 19.6 High-Throughput Screening for Protein-Protein Interactions Using Yeast Two-Hybrid Arrays
    8. UNIT 19.7 Identification of Protein Interactions by Far Western Analysis
    9. UNIT 19.8 Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions
    10. UNIT 19.9 Identifying Protein Interactions by Hydroxyl-Radical Protein Footprinting
    11. UNIT 19.10 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC) Analysis
    12. UNIT 19.11 Production and Use of Trimeric Isoleucine Zipper Fusion Proteins to Study Surface Receptor Ligand Interactions
    13. UNIT 19.12 Fluorescence Quenching Methods to Study Lipid-Protein Interactions
    14. UNIT 19.13 Overview of Biacore Systems and Their Applications
    15. UNIT 19.14 Using Biacore to Measure the Binding Kinetics of an Antibody-Antigen Interaction
    16. UNIT 19.15 Identifying Small-Molecule Modulators of Protein-Protein Interactions
    17. UNIT 19.16 Determination of Protein Contacts by Chemical Cross-Linking With EDC and Mass Spectrometry
    18. UNIT 19.17 Membrane-Based Yeast Two-Hybrid System to Detect Protein Interactions
    19. UNIT 19.18 Detection and Analysis of Protein-Protein Interactions of Organellar and Prokaryotic Proteomes by Blue Native and Colorless Native Gel Electrophoresis
    20. UNIT 19.19 Tandem Affinity Purification of Proteins
    21. UNIT 19.20 Strep/FLAG Tandem Affinity Purification (SF-TAP) to Study Protein Interactions
    22. UNIT 19.21 Identification of Protein-Protein Interactions by Surface Plasmon Resonance followed by Mass Spectrometry
    23. UNIT 19.22 Capture and Qualitative Analysis of the Activated Fc Receptor Complex from Live Cells
    24. UNIT 19.23 BioID: A Screen for Protein-Protein Interactions
    25. UNIT 19.24 Development and Use of IgM/J-Chain Fusion Proteins for Characterization of Immunoglobulin Superfamily Ligand-Receptor Interactions
    26. UNIT 19.25 Measuring Protein-Protein and Protein-Nucleic Acid Interactions by Biolayer Interferometry
    27. UNIT 19.26 Analysis of Protein Ligand-Receptor Binding by Photoaffinity Cross-Linking
    28. UNIT 19.27 Selective Proteomic Proximity Labeling Assay Using Tyramide (SPPLAT): A Quantitative Method for the Proteomic Analysis of Localized Membrane-Bound Protein Clusters
    29. UNIT 19.28 Designing and Applying Proximity-Dependent Hybridization Chain Reaction
    30. UNIT 19.29 Non-Gradient Blue Native Polyacrylamide Gel Electrophoresis
    31. UNIT 19.30 Bioluminescence Resonance Energy Transfer (BRET)-Based Synthetic Sensor Platform for Drug Discovery
  23. Chapter 20 Quantitation of Protein Interactions
    1. Introduction
    2. UNIT 20.1 Overview of the Quantitation of Protein Interactions
    3. UNIT 20.2 Measuring Protein Interactions by Optical Biosensors
    4. UNIT 20.3 Analytical Centrifugation: Equilibrium Approach
    5. UNIT 20.4 Titration Microcalorimetry
    6. UNIT 20.5 Reduced-Scale Large-Zone Analytical Gel-Filtration Chromatography for Measurement of Protein Association Equilibria
    7. UNIT 20.6 Size-Exclusion Chromatography with On-Line Light Scattering
    8. UNIT 20.7 Analytical Ultracentrifugation: Sedimentation Velocity Analysis
    9. UNIT 20.8 Spectroscopic Methods for the Determination of Protein Interactions
    10. UNIT 20.9 Application of Amide Proton Exchange Mass Spectrometry for the Study of Protein-Protein Interactions
    11. UNIT 20.10 Circular Dichroism to Study Protein Interactions
    12. UNIT 20.11 Quantitative Determination of Protein Stability and Ligand Binding by Pulse Proteolysis
    13. UNIT 20.12 Overview of Current Methods in Sedimentation Velocity and Sedimentation Equilibrium Analytical Ultracentrifugation
    14. UNIT 20.13 Thermodynamic Analysis of Weak Protein Interactions Using Sedimentation Equilibrium
  24. Chapter 21 Peptidases
    1. Introduction
    2. UNIT 21.1 Proteases
    3. UNIT 21.2 Papain-like Cysteine Proteases
    4. UNIT 21.3 Overview of Pepsin-like Aspartic Peptidases
    5. UNIT 21.4 Metalloproteases
    6. UNIT 21.5 Purification and Characterization of Proteasomes from Saccharomyces cerevisiae
    7. UNIT 21.6 Purification of the Eukaryotic 20S Proteasome
    8. UNIT 21.7 Serpins (Serine Protease Inhibitors)
    9. UNIT 21.8 Caspases
    10. UNIT 21.9 Use of GFP as a Reporter for the Analysis of Sequence-Specific Proteases
    11. UNIT 21.10 An Overview of Serine Proteases
    12. UNIT 21.11 Over-Expression and Purification of Active Serine Proteases and Their Variants from Escherichia coli Inclusion Bodies
    13. UNIT 21.12 Assaying Proteases in Cellular Environments
    14. UNIT 21.13 Expression, Purification, and Characterization of Caspases
    15. UNIT 21.14 Expression, Purification, and Characterization of Aspartic Endopeptidases: Plasmodium Plasmepsins and “Short” Recombinant Human Pseudocathepsin
    16. UNIT 21.15 Zymography of Metalloproteinases
    17. UNIT 21.16 Handling Metalloproteinases
    18. UNIT 21.17 Applications for Chemical Probes of Proteolytic Activity
    19. UNIT 21.18 Proteomic Identification of Cellular Protease Substrates Using Isobaric Tags for Relative and Absolute Quantification (iTRAQ)
    20. UNIT 21.19 The CLIP-CHIP Oligonucleotide Microarray: Dedicated Array for Analysis of All Protease, Nonproteolytic Homolog, and Inhibitor Gene Transcripts in Human and Mouse
    21. UNIT 21.20 Purification and Characterization of Gingipains
    22. UNIT 21.21 Microplate Assay For Cathepsin Detection in Viable Cells Using Derivatives of 4-Methoxy-β-Naphthylamide
    23. UNIT 21.22 Proteolytic Fingerprinting of Complex Biological Samples Using Combinatorial Libraries of Fluorogenic Probes
  25. Chapter 22 Gel-Based Proteome Analysis
    1. Introduction
    2. UNIT 22.1 Overview of Proteome Analysis
    3. UNIT 22.2 Protein Profiling Using Two-Dimensional Difference Gel Electrophoresis (2-D DIGE)
    4. UNIT 22.3 Laser Capture Microdissection for Proteome Analysis
    5. UNIT 22.4 Preparing Protein Extracts for Quantitative Two-Dimensional Gel Comparison
    6. UNIT 22.5 Isolation of Organelles and Prefractionation of Protein Extracts Using Free-Flow Electrophoresis
    7. UNIT 22.6 Protein Profiling by Microscale Solution Isoelectrofocusing (MicroSol-IEF)
  26. Chapter 23 Non-Gel-Based Proteome Analysis
    1. Introduction
    2. UNIT 23.1 Analysis of Protein Composition Using Multidimensional Chromatography and Mass Spectrometry
    3. UNIT 23.2 The Isotope-Coded Affinity Tag Method for Quantitative Protein Profile Comparison and Relative Quantitation of Cysteine Redox Modifications
    4. UNIT 23.3 Proteomic Analysis Using 2-D Liquid Separations of Intact Proteins From Whole-Cell Lysates
    5. Unit 23.4 Quantitative Protein Analysis Using Enzymatic [18O]Water Labeling
    6. UNIT 23.5 Peptide Extraction from Formalin-Fixed Paraffin-Embedded Tissue
    7. UNIT 23.6 Sample Preparation and In-Solution Protease Digestion of Proteins for Chromatography-Based Proteomic Analysis
    8. UNIT 23.7 Middle-Down and Top-Down Mass Spectrometric Analysis of Co-occurring Histone Modifications
  27. Chapter 24 Targeted Proteomics
    1. Introduction
    2. UNIT 24.1 Human Serum and Plasma Proteomics
    3. UNIT 24.2 Organelle Proteomics
    4. UNIT 24.3 Glycoproteomics Using Chemical Immobilization
    5. UNIT 24.4 Phosphoproteomics
    6. UNIT 24.5 Proteomic Analysis of Protein Deamidation
    7. UNIT 24.6 Using Single Lectins to Enrich Glycoproteins in Conditioned Media
    8. UNIT 24.7 Large-Scale Identification of the Arginine Methylome by Mass Spectrometry
    9. UNIT 24.8 Global Proteomics Analysis of Protein Lysine Methylation
    10. UNIT 24.9 Characterization of Protein Content Present in Exosomes Isolated from Conditioned Media and Urine
    11. UNIT 24.10 Analysis of Protein O-GlcNAcylation by Mass Spectrometry
  28. Chapter 25 Proteome Bioinformatics
    1. Introduction
    2. UNIT 25.1 Bioinformatics Analysis for Interactive Proteomics
    3. UNIT 25.2 Overview of Tandem Mass Spectrometry (MS/MS) Database Search Algorithms
    4. UNIT 25.3 The Publication and Database Deposition of Molecular Interaction Data
    5. UNIT 25.4 PRIDE: Data Submission and Analysis
    6. UNIT 25.5 Using Spectral Libraries for Peptide Identification from Tandem Mass Spectrometry (MS/MS) Data
  29. Chapter 26 Protein Engineering
    1. Introduction
    2. UNIT 26.1 Misincorporation Proton-Alkyl Exchange (MPAX): Engineering Cysteine Probes into Proteins
    3. UNIT 26.2 Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras
    4. UNIT 26.3 Incorporation of Isotopically Enriched Amino Acids
    5. UNIT 26.4 Recombinant Protein Purification by Self-Cleaving Elastin-like Polypeptide Fusion Tag
    6. UNIT 26.5 TimeSTAMP Tagging of Newly Synthesized Proteins
    7. UNIT 26.6 Site-saturation Mutagenesis: A Powerful Tool for Structure-Based Design of Combinatorial Mutation Libraries
    8. UNIT 26.7 Survey of Protein Engineering Strategies
  30. Chapter 27 Protein Arrays
    1. Introduction
    2. UNIT 27.1 Overview of Protein Microarrays
    3. UNIT 27.2 Nucleic Acid Programmable Protein Arrays: Versatile Tools for Array-Based Functional Protein Studies
    4. UNIT 27.3 Protein Microarrays for Identification of Novel Extracellular Protein-Protein Interactions
    5. UNIT 27.4 Preparation of Recombinant Protein Spotted Arrays for Proteome-Wide Identification of Kinase Targets
    6. UNIT 27.5 Detecting Low-Affinity Extracellular Protein Interactions Using Protein Microarrays
    7. UNIT 27.6 Using Antibody Arrays to Measure Protein Abundance and Glycosylation: Considerations for Optimal Performance
    8. UNIT 27.7 Preparation and Use of Reverse Protein Microarrays
    9. UNIT 27.8 Post-Translational Modification Profiling—a High-Content Assay for Identifying Protein Modifications in Mammalian Cellular Systems
  31. Chapter 28 Protein Folding
    1. Introduction
    2. UNIT 28.1 Fast Relaxation Imaging in Living Cells
    3. UNIT 28.2 Overview of Protein Folding Mechanisms: Experimental and Theoretical Approaches to Probing Energy Landscapes
    4. UNIT 28.3 Circular Dichroism in Protein Folding Studies
    5. UNIT 28.4 Determining the Conformational Stability of a Protein from Urea and Thermal Unfolding Curves
    6. UNIT 28.5 Refolding of SDS-Denatured Proteins Using Amphipathic Cosolvents and Osmolytes
    7. Unit 28.6 Overview of the Regulation of Disulfide Bond Formation in Peptide and Protein Folding
    8. Unit 28.7 Chemical Methods for Producing Disulfide Bonds in Peptides and Proteins to Study Folding Regulation
    9. Unit 28.8 Chemical Methods and Approaches to the Regioselective Formation of Multiple Disulfide Bonds
    10. UNIT 28.9 Analysis of Protein Stability and Ligand Interactions by Thermal Shift Assay
    11. UNIT 28.10 Reactivation of Aggregated Proteins by the ClpB/DnaK Bi-Chaperone System
  32. Chapter 29 Membrane Proteins
    1. Introduction
    2. UNIT 29.1 Introduction to Membrane Proteins
    3. UNIT 29.2 Overexpression of Membrane Proteins Using Pichia pastoris
    4. UNIT 29.3 Radioligand Binding Analysis as a Tool for Quality Control of GPCR Production for Structural Characterization: Adenosine A2aR as a Template for Study
    5. UNIT 29.4 Purification of the Human G Protein−Coupled Receptor Adenosine A2aR in a Stable and Functional Form Expressed in Pichia pastoris
    6. UNIT 29.5 HPLC-SEC Characterization of Membrane Protein-Detergent Complexes
    7. UNIT 29.6 High-Throughput Cloning and Expression of Integral Membrane Proteins in Escherichia coli
    8. UNIT 29.7 Methods for Studying Interactions of Detergents and Lipids with α-Helical and β-Barrel Integral Membrane Proteins
    9. UNIT 29.8 Membrane Transport Piece by Piece: Production of Transmembrane Peptides for Structural and Functional Studies
    10. UNIT 29.9 Expression and Purification of Haemophilus influenzae Rhomboid Intramembrane Protease GlpG for Structural Studies
    11. UNIT 29.10 Tetra Detector Analysis of Membrane Proteins
    12. UNIT 29.11 General qPCR and Plate Reader Methods for Rapid Optimization of Membrane Protein Purification and Crystallization Using Thermostability Assays
    13. UNIT 29.12 Detergent Analysis in Protein Samples Using Mid-Infrared (MIR) Spectroscopy
    14. UNIT 29.13 Applications of Lipid Nanodiscs for the Study of Membrane Proteins by Surface Plasmon Resonance
    15. UNIT 29.14 Cell-Free Expression of G Protein–Coupled Receptors
    16. UNIT 29.15 Expression, Solubilization, and Purification of Bacterial Membrane Proteins
    17. UNIT 29.16 In Situ Proteolysis for Crystallization of Membrane Bound Cytochrome P450 17A1 and 17A2 Proteins from Zebrafish
    18. UNIT 29.17 Correlative Förster Resonance Electron Transfer-Proximity Ligation Assay (FRET-PLA) Technique for Studying Interactions Involving Membrane Proteins
    19. UNIT 29.18 Rapid Preparation of a Plasma Membrane Fraction: Western Blot Detection of Translocated Glucose Transporter 4 from Plasma Membrane of Muscle and Adipose Cells and Tissues
  33. Chapter 30 Intracellular Studies
    1. UNIT 30.1 Site-Specific Protein Labeling with SNAP-Tags
    2. UNIT 30.2 Protein Knockouts in Living Eukaryotes Using deGradFP and Green Fluorescent Protein Fusion Targets
    3. UNIT 30.3 Pulse-Chase Analysis for Studying Protein Synthesis and Maturation
    4. UNIT 30.4 Ligand-Directed Profiling of Organelles with Internalizing Phage Libraries
  34. Appendix 1 Useful Data
    1. APPENDIX 1A Characteristics of Amino Acids
    2. APPENDIX 1B Commonly Used Detergents
    3. APPENDIX 1C Conversion Factors and Half-Life Information for Radioactivity
    4. APPENDIX 1D Common Conversion Factors
  35. Appendix 2 Laboratory Guidelines, Equipment, and Stock Solutions
    1. APPENDIX 2A Laboratory Safety
    2. APPENDIX 2B Safe Use of Radioisotopes
    3. APPENDIX 2C Centrifuges and Rotors
    4. APPENDIX 2D Standard Laboratory Equipment
    5. APPENDIX 2E Commonly Used Reagents
  36. Appendix 3 Commonly Used Techniques
    1. APPENDIX 3A Use of Protein Folding Reagents
    2. APPENDIX 3B Dialysis
    3. APPENDIX 3C Techniques for Mammalian Cell Tissue Culture
    4. APPENDIX 3D Importing Biological Materials
    5. APPENDIX 3E Silanizing Glassware
    6. APPENDIX 3F Protein Precipitation Using Ammonium Sulfate
    7. APPENDIX 3G Statistics: Detecting Differences Among Groups
    8. APPENDIX 3H Analyzing Radioligand Binding Data
  37. Appendix 4 Molecular Biology Techniques
    1. APPENDIX 4 Molecular Biology Techniques
    2. APPENDIX 4A Media Preparation and Bacteriological Tools
    3. APPENDIX 4B Growth in Liquid or Solid Media
    4. APPENDIX 4C Preparation of Plasmid DNA
    5. APPENDIX 4D Introduction of Plasmid DNA into Cells
    6. APPENDIX 4E Purification and Concentration of DNA from Aqueous Solutions
    7. APPENDIX 4F Agarose Gel Electrophoresis
    8. APPENDIX 4G Southern Blotting
    9. APPENDIX 4H Hybridization Analysis of DNA Blots
    10. APPENDIX 4I Digestion of DNA with Restriction Endonucleases
    11. APPENDIX 4J The Polymerase Chain Reaction
    12. APPENDIX 4K Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
    13. APPENDIX 4L Growth and Manipulation of Yeast
  38. Appendix 5 Biophysical Methods: Data Analysis
    1. APPENDIX 5A Theoretical Aspects of the Quantitative Characterization of Ligand Binding

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