Current Protocols in Protein Science

Table of Contents

• Foreword
• Preface
• Chapter 1 Strategies of Protein Purification and Characterization
  • Unit 1.1 Overview of Protein Purification and Characterization
  • Unit 1.2 Strategies for Protein Purification
  • Unit 1.3 Protein Purification Flow Charts
  • Unit 1.4 Purification of Glutamate Dehydrogenase from Liver and Brain
  • Unit 1.5 Overview of the Physical State of Proteins within Cells
• Chapter 2 Computational Analysis
  • Introduction
  • Unit 2.1 Computational Methods for Protein Sequence Comparison and Search
  • Unit 2.2 Hydrophobicity Profiles for Protein Sequence Analysis
  • Unit 2.3 Protein Secondary Structure Prediction
  • Unit 2.4 Internet Basics
  • Unit 2.5 Sequence Similarity Searching Using the BLAST Family of Programs
  • Unit 2.6 Protein Databases on the Internet
  • Unit 2.7 Protein Tertiary Structure Prediction
  • Unit 2.8 Protein Tertiary Structure Modeling
  • Unit 2.9 Comparative Protein Structure Modeling Using MODELLER
  • Unit 2.10 Protein Charge Determination
  • Unit 2.11 Basic Protein Sequence Analysis
  • Unit 2.12 Minimotif Miner: A Computational Tool to Investigate Protein Function, Disease, and Genetic Diversity
  • Unit 2.13 Protein Structure Analysis Online
  • Unit 2.14 Protein Structural Domains: Definition and Prediction
• Chapter 3 Detection and Assay Methods
  • Introduction
  • Unit 3.1 Spectrophotometric Determination of Protein Concentration
  • Unit 3.2 Quantitative Amino Acid Analysis
  • Unit 3.3 In Vitro Radiolabeling of Peptides and Proteins
  • Unit 3.4 Assays for Determination of Protein Concentration
  • Unit 3.5 Kinetic Assay Methods
  • Unit 3.6 Protein Biotinylation
  • Unit 3.7 Metabolic Labeling with Amino Acids
  • Unit 3.8 Analysis of Selenocysteine‐Containing Proteins
  • Unit 3.9 Solid‐Phase Profiling of Proteins
  • Unit 3.10 Microvolume Spectrophotometric and Fluorometric Determination of Protein Concentration
• Chapter 4 Extraction, Stabilization, and Concentration
  • Introduction
  • Unit 4.1 Overview of Cell Fractionation
  • Unit 4.2 Purification of Organelles from Mammalian Cells
  • Unit 4.3 Subcellular Fractionation of Tissue Culture Cells
  • Unit 4.4 Desalting, Concentration, and Buffer Exchange by Dialysis and Ultrafiltration
  • Unit 4.5 Selective Precipitation of Proteins
  • Unit 4.6 Long‐Term Storage of Proteins
  • Unit 4.7 Extraction of Proteins from Plant Tissues
  • Unit 4.8 The Use of Detergents to Purify Membrane Proteins
  • Unit 4.9 Trehalose and Protein Stability
• Chapter 5 Production of Recombinant Proteins
  • Introduction
  • Unit 5.1 Production of Recombinant Proteins in Escherichia coli
  • Unit 5.2 Selection of Escherichia coli Expression Systems
  • Unit 5.3 Fermentation and Growth of Escherichia coli for Optimal Protein Production
  • Unit 5.4 Overview of the Baculovirus Expression System
  • Unit 5.5 Protein Expression in the Baculovirus System
  • Unit 5.6 Overview of Protein Expression in Saccharomyces cerevisiae
  • Unit 5.7 Overview of Protein Expression in Pichia pastoris
  • Unit 5.8 Culture of Yeast for the Production of Heterologous Proteins
  • Unit 5.9 Overview of Protein Expression by Mammalian Cells
  • Unit 5.10 Production of Recombinant Proteins in Mammalian Cells
  • Unit 5.11 Overview of the Vaccinia Virus Expression System
  • Unit 5.12 Preparation of Cell Cultures and Vaccinia Virus Stocks
  • Unit 5.13 Generation of Recombinant Vaccinia Viruses
  • Unit 5.14 Characterization of Recombinant Vaccinia Viruses and Their Products
  • Unit 5.15 Gene Expression Using the Vaccinia Virus/ T7 RNA Polymerase Hybrid System
  • Unit 5.16 Choice of Cellular Protein Expression System
  • Unit 5.17 Use of the Gateway System for Protein Expression in Multiple Hosts
  • Unit 5.18 Wheat Germ Cell‐Free Expression System for Protein Production
  • Unit 5.19 Overview on the Expression of Toxic Gene Products in Escherichia coli
  • Unit 5.20 MultiBac: Multigene Baculovirus‐Based Eukaryotic Protein Complex Production
  • Unit 5.21 Recombinant Protein Complex Expression in E. coli
  • Unit 5.22 A Bacterial Cell‐Free Expression System to Produce Membrane Proteins and Proteoliposomes: From cDNA to Functional Assay
  • Unit 5.23 Autoinduction of Protein Expression
  • Unit 5.24 Strategies to Optimize Protein Expression in E. coli
• Chapter 6 Purification of Recombinant Proteins
  • Introduction
  • Unit 6.1 Overview of the Purification of Recombinant Proteins Produced in Escherichia coli
  • Unit 6.2 Preparation of Soluble Proteins from Escherichia coli
  • Unit 6.3 Preparation and Extraction of Insoluble (Inclusion‐Body) Proteins from Escherichia coli
  • Unit 6.4 Overview of Protein Folding
  • Unit 6.5 Folding and Purification of Insoluble (Inclusion Body) Proteins from Escherichia coli
  • Unit 6.6 Expression and Purification of GST Fusion Proteins
  • Unit 6.7 Expression and Purification of Thioredoxin Fusion Proteins
  • Unit 6.8 Automated Large‐Scale Purification of a Recombinant G‐Protein‐Coupled Neurotensin Receptor
  • Unit 6.9 Overview on Concepts and Applications of Fab Antibody Fragments
  • Unit 6.10 E. coli Expression and Purification of Fab Antibody Fragments
  • Unit 6.11 Elastin‐Like Polypeptides as a Purification Tag for Recombinant Proteins
• Chapter 7 Characterization of Recombinant Proteins
  • Introduction
  • Unit 7.1 Overview of the Characterization of Recombinant Proteins
  • Unit 7.2 Determining the Identity and Purity of Recombinant Proteins by UV Absorption Spectroscopy
  • Unit 7.3 Determining the Identity and Structure of Recombinant Proteins
  • Unit 7.4 Transverse Urea‐Gradient Gel Electrophoresis
  • Unit 7.5 Analytical Ultracentrifugation
  • Unit 7.6 Determining the CD Spectrum of a Protein
  • Unit 7.7 Determining the Fluorescence Spectrum of a Protein
  • Unit 7.8 Light Scattering
  • Unit 7.9 Measuring Protein Thermostability by Differential Scanning Calorimetry
  • Unit 7.10 Characterizing Recombinant Proteins Using HPLC Gel Filtration and Mass Spectrometry
  • Unit 7.11 Rapid Screening of E. coli Extracts by Heteronuclear NMR
  • Unit 7.12 Determination of Membrane Protein Molecular Weight Using Sedimentation Equilibrium Analytical Ultracentrifugation
  • Unit 7.13 Methods for the Design and Analysis of Sedimentation Velocity and Sedimentation Equilibrium Experiments with Proteins
• Chapter 8 Conventional Chromatographic Separations
  • Introduction
  • Unit 8.1 Overview of Conventional Chromatography
  • Unit 8.2 Ion‐Exchange Chromatography
  • Unit 8.3 Gel‐Filtration Chromatography
  • Unit 8.4 Hydrophobic‐Interaction Chromatography
  • Unit 8.5 Chromatofocusing
  • Unit 8.6 Hydroxylapatite Chromatography
  • Unit 8.7 Reversed‐Phase High Performance Liquid Chromatography of Proteins
  • Unit 8.8 Expanded‐Bed Adsorption Chromatography
  • Unit 8.9 Displacement Chromatography of Proteins
• Chapter 9 Affinity Purification
  • Introduction
  • Unit 9.1 Lectin Affinity Chromatography
  • Unit 9.2 Dye Affinity Chromatography
  • Unit 9.3 Affinity Purification of Natural Ligands
  • Unit 9.4 Metal‐Chelate Affinity Chromatography
  • Unit 9.5 Immunoaffinity Chromatography
  • Unit 9.6 Purification of Sequence‐Specific DNA‐Binding Proteins by Affinity Chromatography
  • Unit 9.7 Purification of DNA‐Binding Proteins Using Biotin/Streptavidin Affinity Systems
  • Unit 9.8 Immunoprecipitation
  • Unit 9.9 Overview of Affinity Tags for Protein Purification
• Chapter 10 Electrophoresis
  • Introduction
  • Unit 10.1 One‐Dimensional SDS Gel Electrophoresis of Proteins
  • Unit 10.2 One‐Dimensional Isoelectric Focusing of Proteins in Slab Gels
  • Unit 10.3 One‐Dimensional Electrophoresis Using Nondenaturing Conditions
  • Unit 10.4 Two‐Dimensional Gel Electrophoresis
  • Unit 10.5 Protein Detection in Gels Using Fixation
  • Unit 10.6 Protein Detection in Gels Without Fixation
  • Unit 10.7 Electroblotting from Polyacrylamide Gels
  • Unit 10.8 Detection of Proteins on Blot Membranes
  • Unit 10.9 Capillary Electrophoresis of Proteins and Peptides
  • Unit 10.10 Immunoblot Detection
  • Unit 10.11 Autoradiography
  • Unit 10.12 Overview of Digital Electrophoresis Analysis
  • Unit 10.13 Capillary Electrophoresis of Peptides and Proteins Using Isoelectric Buffers
• Chapter 11 Chemical Analysis
  • Introduction
  • Unit 11.1 Enzymatic Digestion of Proteins in Solution
  • Unit 11.2 Enzymatic Digestion of Proteins on PVDF Membranes
  • Unit 11.3 Enzymatic Digestion of Proteins in Gels for Mass Spectrometric Identification and Structural Analysis
  • Unit 11.4 Chemical Cleavage of Proteins in Solution
  • Unit 11.5 Chemical Cleavage of Proteins on Membranes
  • Unit 11.6 Reversed‐Phase Isolation of Peptides
  • Unit 11.7 Removal of N‐Terminal Blocking Groups from Proteins
  • Unit 11.8 C‐Terminal Sequence Analysis
  • Unit 11.9 Amino Acid Analysis
  • Unit 11.10 N‐Terminal Sequence Analysis of Proteins and Peptides
  • Unit 11.11 Determination of Disulfide‐Bond Linkages in Proteins
• Chapter 12 Post‐Translational Modification: Glycosylation
  • Introduction
  • Unit 12.1 Overview of Glycoconjugate Analysis
  • Unit 12.2 Metabolic Radiolabeling of Animal Cell Glycoconjugates
  • Unit 12.3 Inhibition of N‐Linked Glycosylation
  • Unit 12.4 Endoglycosidase and Glycoamidase Release of N‐Linked Glycans
  • Unit 12.5 Detection of Glycophospholipid Anchors on Proteins
  • Unit 12.6 Determining the Structure of Oligosaccharides N‐ and O‐Linked to Glycoproteins
  • Unit 12.7 Determining the Structure of Glycan Moieties by Mass Spectrometry
  • Unit 12.8 Detection and Analysis of Proteins Modified by O‐Linked N‐Acetylglucosamine
  • Unit 12.9 High‐Throughput Lectin Microarray‐Based Analysis of Live Cell Surface Glycosylation
  • Unit 12.10 Preparation and Analysis of Glycan Microarrays
• Chapter 13 Post‐Translational Modification: Phosphorylation and Phosphatases
  • Introduction
  • Unit 13.1 Overview of Protein Phosphorylation
  • Unit 13.2 Labeling Cultured Cells with 32Pi and Preparing Cell Lysates for Immunoprecipitation
  • Unit 13.3 Phosphoamino Acid Analysis
  • Unit 13.4 Detection of Phosphorylation by Immunological Techniques
  • Unit 13.5 Detection of Phosphorylation by Enzymatic Techniques
  • Unit 13.6 Preparation and Application of Polyclonal and Monoclonal Sequence‐Specific Anti‐Phosphoamino Acid Antibodies
  • Unit 13.7 Assays of Protein Kinases Using Exogenous Substrates
  • Unit 13.8 Permeabilization Strategies to Study Protein Phosphorylation
  • Unit 13.9 Phosphopeptide Mapping and Identification of Phosphorylation Sites
  • Unit 13.10 Use of Protein Phosphatase Inhibitors
  • Unit 13.11 Tyrosine Phosphorylation Enrichment and Subsequent Analysis by MALDI‐TOF/TOF MS/MS and LC‐ESI‐IT‐MS/MS
  • Unit 13.12 In‐Gel Phosphatase Assay Using Fluorogenic and Radioactive Substrates
• Chapter 14 Post‐Translational Modification: Specialized Applications
  • Introduction
  • Unit 14.1 Analysis of Disulfide Bond Formation
  • Unit 14.2 Analysis of Protein Acylation
  • Unit 14.3 Analysis of Protein Prenylation In Vitro and In Vivo Using Functionalized Phosphoisoprenoids
  • Unit 14.4 Analysis of Oxidative Modification of Proteins
  • Unit 14.5 Analysis of Protein Ubiquitination
  • Unit 14.6 Analysis of Protein S‐Nitrosylation
  • Unit 14.7 Tyrosine O‐Sulfation
  • Unit 14.8 Analysis of Protein Sumoylation
  • Unit 14.9 Identifying and Quantifying Sites of Protein Methylation by Heavy Methyl SILAC
  • Unit 14.10 Analysis of Histone Modifications by Mass Spectrometry
  • Unit 14.11 Analysis of Protein Lysine Acetylation In Vitro and In Vivo
  • Unit 14.12 Determination of Protein Lysine Deacetylation
• Chapter 15 Chemical Modification of Proteins
  • Introduction
  • Unit 15.1 Modification of Cysteine
  • Unit 15.2 Modification of Amino Groups
  • Unit 15.3 Site‐Specific Protein Labeling via Sortase‐Mediated Transpeptidation
• Chapter 16 Mass Spectrometry
  • Introduction
  • Unit 16.1 Overview of Peptide and Protein Analysis by Mass Spectrometry
  • Unit 16.2 Matrix‐Assisted Laser Desorption/Ionization Time‐of‐Flight Mass Analysis of Peptides
  • Unit 16.3 Sample Preparation for MALDI Mass Analysis of Peptides and Proteins
  • Unit 16.4 In‐Gel Digestion of Proteins for MALDI‐MS Fingerprint Mapping
  • Unit 16.5 Searching Sequence Databases Over the Internet: Protein Identification Using MS‐Fit
  • Unit 16.6 Searching Sequence Databases Over the Internet: Protein Identification Using MS‐Tag
  • Unit 16.7 Enzymatic Approaches for Obtaining Amino Acid Sequence: On‐Target Ladder Sequencing
  • Unit 16.8 Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using a Nanospray Interface
  • Unit 16.9 Introducing Samples Directly into Electrospray Ionization Mass Spectrometers Using Microscale Capillary Liquid Chromatography
  • Unit 16.10 Protein Identification Using a Quadrupole Ion Trap Mass Spectrometer and SEQUEST Database Matching
  • Unit 16.11 De Novo Peptide Sequencing via Manual Interpretation of MS/MS Spectra
  • Unit 16.12 Rapid Detergent Removal from Peptide Samples with Ethyl Acetate for Mass Spectrometry Analysis
  • Unit 16.13 Preparation and Analysis of Proteins and Peptides Using MALDI TOF/TOF Mass Spectrometry
• Chapter 17 Structural Biology
  • Introduction
  • Unit 17.1 Overview of Protein Structural and Functional Folds
  • Unit 17.2 Electron Microscopy and Image Processing: An Essential Tool for Structural Analysis of Macromolecules
  • Unit 17.3 Principles of Macromolecular X‐Ray Crystallography
  • Unit 17.4 Crystallization of Macromolecules
  • Unit 17.5 Overview on the Use of NMR to Examine Protein Structure
  • Unit 17.6 Investigating Solution‐Phase Protein Structure and Dynamics by Hydrogen Exchange Mass Spectrometry
  • Unit 17.7 Introduction to Atomic Force Microscopy (AFM) in Biology
  • Unit 17.8 Raman Spectroscopy of Proteins
  • Unit 17.9 Crystallization of Integral Membrane Proteins
  • Unit 17.10 Application of Dynamic Light Scattering in Protein Crystallization
  • Unit 17.11 The STINT‐NMR Method for Studying In‐cell Protein‐Protein Interactions
  • Unit 17.12 Native Mass Spectrometry as a Tool in Structural Biology
  • Unit 17.13 Cryo‐Electron Tomography for Structural Characterization of Macromolecular Complexes
• Chapter 18 Preparation and Handling of Peptides
  • Introduction
  • Unit 18.1 Introduction to Peptide Synthesis
  • Unit 18.2 Combinatorial Peptide Synthesis on a Microchip
  • Unit 18.3 Synthetic Peptides for Production of Antibodies that Recognize Intact Proteins
  • Unit 18.4 Native Chemical Ligation of Polypeptides
  • Unit 18.5 Synthesis and Application of Peptide Dendrimers As Protein Mimetics
  • Unit 18.6 Disulfide Bond Formation in Peptides
  • Unit 18.7 Guide for Resin and Linker Selection in Solid‐Phase Peptide Synthesis
  • Unit 18.8 Overview of Solid Phase Synthesis of “Difficult Peptide” Sequences
  • Unit 18.9 Phage Display for Generating Peptide Reagents
  • Unit 18.10 Utilizing Peptide SPOT Arrays to Identify Protein Interactions
  • Unit 18.11 Expression and Purification of ZEBRA Fusion Proteins and Applications for the Delivery of Macromolecules into Mammalian Cells
  • Unit 18.12 Storage and Handling Guidelines for Custom Peptides
  • Unit 18.13 Peptidomic Approaches to Study Proteolytic Activity
• Chapter 19 Identification of Protein Interactions
  • Introduction
  • Unit 19.1 Analysis of Protein‐Protein Interactions
  • Unit 19.2 Interaction Trap/Two‐Hybrid System to Identify Interacting Proteins
  • Unit 19.3 Phage‐Based Expression Cloning to Identify Interacting Proteins
  • Unit 19.4 Detection of Protein‐Protein Interactions by Coprecipitation
  • Unit 19.5 Imaging Protein‐Protein Interactions by Förster Resonance Energy Transfer (FRET) Microscopy in Live Cells
  • Unit 19.6 High‐Throughput Screening for Protein‐Protein Interactions Using Yeast Two‐Hybrid Arrays
  • Unit 19.7 Identification of Protein Interactions by Far Western Analysis
  • Unit 19.8 Scintillation Proximity Assay (SPA) Technology to Study Biomolecular Interactions
  • Unit 19.9 Identifying Protein Interactions by Hydroxyl‐Radical Protein Footprinting
  • Unit 19.10 Visualization of Protein Interactions in Living Cells Using Bimolecular Fluorescence Complementation (BiFC) Analysis
  • Unit 19.11 Production and Use of Trimeric Isoleucine Zipper Fusion Proteins to Study Surface Receptor Ligand Interactions
  • Unit 19.12 Fluorescence Quenching Methods to Study Lipid‐Protein Interactions
  • Unit 19.13 Overview of Biacore Systems and Their Applications
  • Unit 19.14 Using Biacore to Measure the Binding Kinetics of an Antibody‐Antigen Interaction
  • Unit 19.15 Identifying Small‐Molecule Modulators of Protein‐Protein Interactions
  • Unit 19.16 Determination of Protein Contacts by Chemical Cross‐Linking With EDC and Mass Spectrometry
  • Unit 19.17 Membrane‐Based Yeast Two‐Hybrid System to Detect Protein Interactions
  • Unit 19.18 Detection and Analysis of Protein‐Protein Interactions of Organellar and Prokaryotic Proteomes by Blue Native and Colorless Native Gel Electrophoresis
  • Unit 19.19 Tandem Affinity Purification of Proteins
  • Unit 19.20 Strep/FLAG Tandem Affinity Purification (SF‐TAP) to Study Protein Interactions
  • Unit 19.21 Identification of Protein‐Protein Interactions by Surface Plasmon Resonance followed by Mass Spectrometry
  • Unit 19.22 Capture and Qualitative Analysis of the Activated Fc Receptor Complex from Live Cells
• Chapter 20 Quantitation of Protein Interactions
  • Introduction
  • Unit 20.1 Overview of the Quantitation of Protein Interactions
  • Unit 20.2 Measuring Protein Interactions by Optical Biosensors
  • Unit 20.3 Analytical Centrifugation: Equilibrium Approach
  • Unit 20.4 Titration Microcalorimetry
  • Unit 20.5 Reduced‐Scale Large‐Zone Analytical Gel‐Filtration Chromatography for Measurement of Protein Association Equilibria
  • Unit 20.6 Size‐Exclusion Chromatography with On‐Line Light Scattering
  • Unit 20.7 Analytical Ultracentrifugation: Sedimentation Velocity Analysis
  • Unit 20.8 Spectroscopic Methods for the Determination of Protein Interactions
  • Unit 20.9 Application of Amide Proton Exchange Mass Spectrometry for the Study of Protein‐Protein Interactions
  • Unit 20.10 Circular Dichroism to Study Protein Interactions
  • Unit 20.11 Quantitative Determination of Protein Stability and Ligand Binding by Pulse Proteolysis
• Chapter 21 Peptidases
  • Introduction
  • Unit 21.1 Proteases
  • Unit 21.2 Papain‐like Cysteine Proteases
  • Unit 21.3 Overview of Pepsin‐like Aspartic Peptidases
  • Unit 21.4 Metalloproteases
  • Unit 21.5 Purification and Characterization of Proteasomes from Saccharomyces cerevisiae
  • Unit 21.6 Purification of the Eukaryotic 20S Proteasome
  • Unit 21.7 Serpins (Serine Protease Inhibitors)
  • Unit 21.8 Caspases
  • Unit 21.9 Use of GFP as a Reporter for the Analysis of Sequence‐Specific Proteases
  • Unit 21.10 An Overview of Serine Proteases
  • Unit 21.11 Over‐Expression and Purification of Active Serine Proteases and Their Variants from Escherichia coli Inclusion Bodies
  • Unit 21.12 Assaying Proteases in Cellular Environments
  • Unit 21.13 Expression, Purification, and Characterization of Caspases
  • Unit 21.14 Expression, Purification, and Characterization of Aspartic Endopeptidases: Plasmodium Plasmepsins and “Short” Recombinant Human Pseudocathepsin
  • Unit 21.15 Zymography of Metalloproteinases
  • Unit 21.16 Monitoring Metalloproteinase Activity Using Synthetic Fluorogenic Substrates
  • Unit 21.17 Applications for Chemical Probes of Proteolytic Activity
  • Unit 21.18 Proteomic Identification of Cellular Protease Substrates Using Isobaric Tags for Relative and Absolute Quantification (iTRAQ)
  • 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
  • Unit 21.20 Purification and Characterization of Gingipains
  • Unit 21.21 Microplate Assay For Cathepsin Detection in Viable Cells Using Derivatives of 4‐Methoxy‐β‐Naphthylamide
• Chapter 22 Gel‐Based Proteome Analysis
  • Introduction
  • Unit 22.1 Overview of Proteome Analysis
  • Unit 22.2 Protein Profiling Using Two‐Dimensional Difference Gel Electrophoresis (2‐D DIGE)
  • Unit 22.3 Laser Capture Microdissection for Proteome Analysis
  • Unit 22.4 Preparing Protein Extracts for Quantitative Two‐Dimensional Gel Comparison
  • Unit 22.5 Isolation of Organelles and Prefractionation of Protein Extracts Using Free‐Flow Electrophoresis
  • Unit 22.6 Protein Profiling by Microscale Solution Isoelectrofocusing (MicroSol‐IEF)
• Chapter 23 Non‐Gel‐Based Proteome Analysis
  • Introduction
  • Unit 23.1 Analysis of Protein Composition Using Multidimensional Chromatography and Mass Spectrometry
  • Unit 23.2 Quantitative Protein Profile Comparisons Using the Isotope‐Coded Affinity Tag Method
  • Unit 23.3 Proteomic Analysis Using 2‐D Liquid Separations of Intact Proteins From Whole‐Cell Lysates
  • Unit 23.4 Quantitative Protein Analysis Using Proteolytic [18O]Water Labeling
  • Unit 23.5 Peptide Extraction from Formalin‐Fixed Paraffin‐Embedded Tissue
  • Unit 23.6 Sample Preparation and In‐Solution Protease Digestion of Proteins for Chromatography‐Based Proteomic Analysis
• Chapter 24 Targeted Proteomics
  • Introduction
  • Unit 24.1 Human Serum and Plasma Proteomics
  • Unit 24.2 Organelle Proteomics
  • Unit 24.3 Glycoproteomics Using Chemical Immobilization
  • Unit 24.4 Phosphoproteomics
• Chapter 25 Proteome Bioinformatics
  • Introduction
  • Unit 25.1 Bioinformatics Analysis for Interactive Proteomics
  • Unit 25.2 Overview of Tandem Mass Spectrometry (MS/MS) Database Search Algorithms
  • Unit 25.3 The Publication and Database Deposition of Molecular Interaction Data
  • Unit 25.4 PRIDE: Data Submission and Analysis
  • Unit 25.5 Using Spectral Libraries for Peptide Identification from Tandem Mass Spectrometry (MS/MS) Data
• Chapter 26 Protein Engineering
  • Introduction
  • Unit 26.1 Misincorporation Proton‐Alkyl Exchange (MPAX): Engineering Cysteine Probes into Proteins
  • Unit 26.2 Combinatorial Recombination of Gene Fragments to Construct a Library of Chimeras
  • Unit 26.3 Incorporation of Isotopically Enriched Amino Acids
  • Unit 26.4 Recombinant Protein Purification by Self‐Cleaving Elastin‐like Polypeptide Fusion Tag
  • Unit 26.5 TimeSTAMP Tagging of Newly Synthesized Proteins
  • Unit 26.6 Site‐saturation Mutagenesis: A Powerful Tool for Structure‐Based Design of Combinatorial Mutation Libraries
  • Unit 26.7 Survey of Protein Engineering Strategies
• Chapter 27 Protein Arrays
  • Introduction
  • Unit 27.2 Nucleic Acid Programmable Protein Arrays: Versatile Tools for Array‐Based Functional Protein Studies
• Chapter 28 Protein Folding
  • Introduction
  • Unit 28.1 Fast Relaxation Imaging in Living Cells
• Chapter 29 Membrane Proteins
  • Unit 29.1 Introduction to Membrane Proteins
  • Unit 29.2 Overexpression of Membrane Proteins Using Pichia pastoris
  • 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
  • Unit 29.4 Purification of the Human G Protein−Coupled Receptor Adenosine A2aR in a Stable and Functional Form Expressed in Pichia pastoris
• Appendix 1 Useful Data
  • Appendix 1A Characteristics of Amino Acids
  • Appendix 1B Commonly Used Detergents
  • Appendix 1C Conversion Factors and Half‐Life Information for Radioactivity
  • Appendix 1D Common Conversion Factors
• Appendix 2 Laboratory Guidelines, Equipment, and Stock Solutions
  • Appendix 2A Laboratory Safety
  • Appendix 2B Safe Use of Radioisotopes
  • Appendix 2C Centrifuges and Rotors
  • Appendix 2D Standard Laboratory Equipment
  • Appendix 2E Commonly Used Reagents
• Appendix 3 Commonly Used Techniques
  • Appendix 3A Use of Protein Folding Reagents
  • Appendix 3B Dialysis
  • Appendix 3C Techniques for Mammalian Cell Tissue Culture
  • Appendix 3D Importing Biological Materials
  • Appendix 3E Silanizing Glassware
  • Appendix 3F Protein Precipitation Using Ammonium Sulfate
  • Appendix 3G Statistics: Detecting Differences Among Groups
  • Appendix 3H Analyzing Radioligand Binding Data
• Appendix 4 Molecular Biology Techniques
  • Appendix 4A Media Preparation and Bacteriological Tools
  • Appendix 4B Growth in Liquid or Solid Media
  • Appendix 4C Preparation of Plasmid DNA
  • Appendix 4D Introduction of Plasmid DNA into Cells
  • Appendix 4E Purification and Concentration of DNA from Aqueous Solutions
  • Appendix 4F Agarose Gel Electrophoresis
  • Appendix 4G Southern Blotting
  • Appendix 4H Hybridization Analysis of DNA Blots
  • Appendix 4I Digestion of DNA with Restriction Endonucleases
  • Appendix 4J The Polymerase Chain Reaction
  • Appendix 4K Quantitation of DNA and RNA with Absorption and Fluorescence Spectroscopy
  • Appendix 4L Growth and Manipulation of Yeast
• Appendix 5 Biophysical Methods: Data Analysis
  • Appendix 5A Theoretical Aspects of the Quantitative Characterization of Ligand Binding
• Appendix Suppliers
  • Selected Suppliers of Reagents and Equipment

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