Proteostasis: Methods and Protocols (Methods in Molecular Biology, 1449)

✍ Scribed by Rune Matthiesen (editor)

• Publisher: Humana
• Year: 2016
• Tongue: English
• Leaves: 490
• Category: Library

✦ Synopsis

This volume highlights the role of proteostasis in human health and associated disease model systems, reflecting its rising importance which has led to the development of new technologies to obtain insight into underling protein mechanistic events. 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.

Authoritative and cutting-edge, Proteostasis: Methods and Protocols aims to become a reference book on proteostasis in human health.

✦ Table of Contents

Preface
Contents
Contributors
Chapter 1: UPS Activation in the Battle Against Aging and Aggregation-Related Diseases: An Extended Review
1 Aging and Aggregation-Related Diseases
1.1 Aging/Models of Aging
1.2 Aggregation-
1.2.1 Alzheimer’s Disease
1.2.2 Parkinson’s Disease
1.2.3 Huntington’s Disease
1.2.4 Amyotrophic Lateral Sclerosis
1.2.5 Prion Diseases
1.3 Proteostasis in Normal Aging and Aggregation-Related Diseases
2 Introduction: The Ubiquitin System
2.1 E1, E2, E3 Enzymes
2.2 Deubiquitinases
3 Introduction: The Proteasome System
3.1 20S Core Proteasome: Structure, Assembly, and Localization
3.1.1 Structure
3.1.2 Assembly
3.1.3 Localization
3.2 26S Proteasome: Structure and Assembly
3.2.1 Structure
3.2.2 Assembly
3.3 Various Proteasome Forms
3.3.1 Immuno-proteasome
3.3.2 Hybrid Proteasomes
3.3.3 Thymo-proteasomes
3.3.4 Other Forms of Proteasomes
3.4 Regulation of the Proteasome Expression and Function
3.4.1 Transcriptional Regulation
3.4.2 Posttranslational Modifications
3.5 Elimination of Proteasomes
4 Proteasome Status During Aging/Senescence in Cellular and Organismal Models
4.1 Cellular Senescence
4.2 Model Organisms
4.2.1 Saccharomyces cerevisiae
4.2.2 Caenorhabditis elegans
4.2.3 Drosophila melanogaster
4.2.4 Rodents
4.2.5 Homo sapiens
5 Proteasome Impairment During Aggregation-Related Diseases
5.1 Alzheimer’s Disease
5.2 Parkinson’s Disease
5.3 Huntington’s Disease
5.4 Amyotrophic Lateral Sclerosis
5.5 Prion Diseases
6 Proteasome Activation During Aging
6.1 Saccharomyces cerevisiae
6.1.1 20S and 19S Proteasome Subunits and Other Proteasome Activators
6.1.2 E1, E2, and E3 Ligases
6.1.3 Deubiquitinases
6.1.4 Other Conditions and Compounds
6.2 Caenorhabditis elegans
6.2.1 20S and 19S Proteasome Subunits
6.2.2 E1, E2, and E3 Ligases
6.2.3 Deubiquitinases
6.2.4 Other Conditions and Compounds
6.3 Drosophila melanogaster
6.3.1 20S and 19S Proteasome Subunits
6.3.2 E1, E2, and E3 Ligases
6.3.3 Deubiquitinases
6.3.4 Other Conditions and Compounds
6.4 Rodents
6.4.1 20S and 19S Proteasome Subunits and Other Proteasome Activators and Components
6.4.2 Other Conditions and Compounds
6.5 Mammalian Cells
6.5.1 20S and 19S Proteasome Subunits and Other Proteasome Activators and Components
6.5.2 E1, E2, and E3 Ligases
6.5.3 Deubiquitinases
6.5.4 Other Conditions and Compounds
6.6 Homo sapiens
6.6.1 Other Conditions and Compounds
7 Proteasome Activation During Aggregation-Related Diseases
7.1 Alzheimer’s Disease (AD)
7.1.1 20S and 19S Proteasome Subunits
7.1.2 E1, E2, and E3 Ligases
7.1.3 Deubiquitinases
7.1.4 Other Conditions and Compounds
7.2 Parkinson’s Disease (PD)
7.2.1 20S and 19S Proteasome Subunits
7.2.2 E1, E2, and E3 Ligases and Ubiquitin
7.2.3 Other Conditions and Compounds
7.3 Huntington’s Disease (HD)
7.3.1 20S and 19S Proteasome Subunits
7.3.2 E1, E2, and E3 Ligases
7.3.3 Deubiquitinases
7.3.4 Other Conditions and Compounds
7.4 Amyotrophic Lateral Sclerosis (ALS)
7.4.1 E1, E2, and E3 Ligases
7.4.2 Other Conditions and Compounds
7.5 Prion Diseases
7.5.1 E1, E2, and E3 Ligases
7.5.2 Other Conditions and Compounds
8 Concluding Remarks
References
Chapter 2: Review and Literature Mining on Proteostasis Factors and Cancer
1 Introduction
2 Materials
3 Methods
3.1 Obtaining Text Corpus from PubMed
3.2 Wordclouds
3.3 Gene Associations with Diseases
3.4 Discussion of Results
4 Notes
References
Chapter 3: Combining Zebrafish and Mouse Models to Test the Function of Deubiquitinating Enzyme (Dubs) Genes in Development: Role of USP45 in the Retina
1 Introduction
2 Materials and Solutions
2.1 Dissection of Mouse Retinas and Preparation of Mouse Eye Sections
2.2 RNA Isolation, cDNA Synthesis, qPCR, and PCR
2.3 In Situ Hybridization on Mouse and Zebrafish Retinal Cryosections
2.4 Zebrafish Embryo Collection, Handling and Fixation, and Morpholino Microinjection
2.5 Solutions
3 Methods
3.1 Dissection of Mouse Retinas for RNA Isolation
3.2 Dissection of Mouse Retinas for Cryosections
3.2.1 Sucrose Embedding
3.2.2 Acrylamide Embedding
3.3 Cloning of the Riboprobe
3.4 Generation of the Riboprobe
3.5 In Situ Hybridization on Mouse Cryosections
3.5.1 Day 1
3.5.2 Day 2
3.5.3 Day 3
3.6 Zebrafish Embryo Collection, Handling and Fixation
3.7 Semi-
3.8 In Situ Hybridization on Zebrafish Retinal Sections
3.9 Morpholino Microinjection in Zebrafish Embryos
3.10 Hematoxylin/Eosin Staining of Zebrafish Retinal Sections
4 Notes
References
Chapter 4: Immunodepletion and Immunopurification as Approaches for CSN Research
1 Introduction
2 Materials
2.1 Production of Immunoaffinity Columns
2.2 Cells and TCE Preparation
2.3 Preparation of CDP
2.4 Eluting the Immunoaffinity Purified CSN Complex
2.5 Clean-Up of FPLC
2.6 Evaluation of immunodepletion
2.7 Deneddylation Activity Assay
3 Methods
3.1 The Production of Affinity Columns
3.2 Preparation of TCE
3.3 Preparation of CDP
3.4 Eluting the Immunoaffinity Purified CSN Complex
3.5 Clean-Up of FPLC
3.6 Evaluating CSN Immunodepletion
3.7 Deneddylation Activity Assay
3.8 Assessment of CSN Interactions
4 Notes
References
Chapter 5: Studying Protein Ubiquitylation in Yeast
1 Introduction
2 Materials
2.1 Components Required for the Visualization of Ubiquitylation on Crude Extracts (Subheading 3.1)
2.2 Components Required for the Purification of Ubiquitin Conjugates Using PolyHis-Tagged Ubiquitin (Subheading 3.2)
2.3 Components Required for Immuno-precipitation in Denaturing Conditions (Subheading 3.3)
2.4 Components Required for the Tandem Purification of a Protein in Denaturing Conditions for the Identification of Ubiquitin-Conjugated Sites (Subheading 3.4)
3 Methods
3.1 Obtaining Evidence of Ubiquitylation on Crude Extracts
3.2 Purification of Ubiquitin Conjugates Using PolyHis-­Tagged Ubiquitin
3.3 Assessment of Protein Ubiquitylation After Immuno-precipitation in Denaturing Conditions
3.4 Tandem Purification of a Protein in Denaturing Conditions for the Identification of Ubiquitin-Conjugated Sites
3.5 Identification of Ubiquitylated Sites Within a Protein of Interest by a Genetic Approach
3.6 Studying the Topology of Ubiquitin Modification
4 Notes
References
Chapter 6: Strategies to Detect Endogenous Ubiquitination of a Target Mammalian Protein
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Reagents and Antibodies
2.3 DELFIA (Perkin Elmer)
2.4 EnVision Instrument (Perkin Elmer)
3 Methods
3.1 Analysis by IP of a Specific Substrate (i.e., EGFR) and Western Blot Anti-ubiquitin
3.1.1 Cell Lysis and IP Reaction
3.1.2 Western Blot Anti-Ub
3.2 Immuno­precipitation of Ub-Modified Proteins from Cellular Lysate
3.2.1 IP Anti-Ub
3.3 ELISA Assays for EGFR Ubiquitination
3.3.1 Plate Coating, Incubation, and Detection
4 Notes
References
Chapter 7: In Vitro Ubiquitination: Self-Ubiquitination, Chain Formation, and Substrate Ubiquitination Assays
1 Introduction
2 Materials
2.1 Buffers and Solutions
2.2 Competent Cells for Protein Expression
2.3 Reagents
3 Methods
3.1 Protein Production
3.1.1 GST Fusion Protein
3.1.2 His-Fusion Protein Production
3.1.3 Untagged Ub Production
3.2 Protein Purification
3.3 In Vitro Ubiquitination Assays
3.3.1 Self-Ubiquitination Assay and Ub Chains Formation
3.3.2 Western Blot Anti-Ub
3.3.3 Substrate Ubiquitination
4 Notes
References
Chapter 8: Isolation of the Ubiquitin-Proteome from Tumor Cell Lines and Primary Cells Using TUBEs
1 Introduction
2 Materials
2.1 Coupling TUBEs/GST to Glutathione Beads
2.2 Cell Lysis and Sample Clearance
2.3 Capturing and Elution of Proteins
2.4 Sample Concentration and Gel Electrophoresis
3 Methods
3.1 Coupling TUBEs/GST to Glutathione Beads: Covalent Cross-Linking
3.2 Cell Lysis and Sample Clearance
3.3 Capturing of Ubiquitylated Proteins by Affinity Chromatography and Elution
3.4 Sample Concentration and Gel Electrophoresis
4 Notes
References
Chapter 9: TUBEs-Mass Spectrometry for Identification and Analysis of the Ubiquitin-Proteome
1 Introduction
2 Materials
2.1 Sypro Ruby Gel Staining and Image Acquisition
2.2 Digestion
2.3 MS Analysis
3 Methods
3.1 Gel Staining and Image Acquisition
3.2 Gel Cut and Digestion of the Gel Slices
3.3 MS Analysis of the Samples
3.4 MS Data Analysis
3.5 Statistical Analysis
3.6 Meta-Analysis
4 Notes
References
Chapter 10: Isolation of Ubiquitinated Proteins to High Purity from In Vivo Samples
1 Introduction
1.1 The bioUb Purification Strategy
1.2 The GFP-
2 Materials
2.1 The bioUb Purification Strategy
2.2 The GFP-
3 Methods
3.1 The bioUb Purification Strategy
3.2 The GFP-
4 Notes
References
Chapter 11: Method for the Purification of Endogenous Unanchored Polyubiquitin Chains
1 Introduction
2 Materials
2.1 Production of GST-ZnF_UBP
2.2 Covalent Immobilization of ZnF_UBP Domain to Cyanogen Bromide-­Activated Sepharose 4B
2.3 Preparation of Protein Extracts for Unanchored Polyubiquitin Chain Purification
2.4 Commercially Available Ubiquitin Antibodies to Probe Purified Unanchored Polyubiquitin Samples
2.5 Deubiquitination Assay
3 Methods
3.1 Generating the FUBE
3.1.1 Cloning the ZnF_UBP Domain of USP5
3.1.2 Over-expression and Purification of ZnF_UBP Domain
3.1.3 Immobilization of Purified ZnF_UBP Domain onto CNBr-­Sepharose
3.2 Preparation of Protein Extracts for the Purification of Unanchored Polyubiquitin Chains
3.3 Affinity Purification of Unanchored Polyubiquitin Chains
3.4 Visualizing the Purified Unanchored Polyubiquitin Chain Pool
3.4.1 Probing the Linkage Profile of FUBE-Purified Unanchored Polyubiquitin Chains
3.4.2 Deubiquitination Assay
3.4.3 Mass Spectrometry-­Based Characterization of Purified Unanchored Polyubiquitin Chains
4 Notes
References
Chapter 12: Fluorescent Tools for In Vivo Studies on the Ubiquitin-­Proteasome System
1 Introduction
2 Materials
2.1 Transgenic C. elegans
2.2 Agarose (Fischer Scientific)
2.3 Glass Slides and Cover Slips (Thermo Scientific)
2.4 Levamisole Hydrochloride (Sigma-Aldrich)
2.5 Microscopes
3 Methods
3.1 Agarose Pads
3.2 Transgenic C. elegans Maintenance
3.3 Live Imaging
4 Notes
References
Chapter 13: Bimolecular Fluorescence Complementation to Assay the Interactions of Ubiquitylation Enzymes in Living Yeast Cells
1 Introduction
1.1 Critical Considerations and Design of BiFC Experiments in Yeast
1.1.1 Advantages and Limitations of BiFC
1.1.2 Choice of Fluorescent Protein Fragments
1.1.3 Construction of Yeast Strains for BiFC Experiments
1.1.4 Negative Controls
2 Materials
2.1 Yeast Cultures
2.2 Microscopy
2.3 Image Processing
3 Methods
3.1 Cell Preparation for Microscopy
3.2 Image Acquisition
3.3 Image Processing and BiFC Signal Quantification
3.3.1 Image Segmentation
3.3.2 Background Subtraction
3.3.3 Autofluorescence Subtraction
3.3.4 BiFC Fluorescence and Cell Property Quantification
4 Notes
References
Chapter 14: Monitoring Ubiquitin-Coated Bacteria via Confocal Microscopy
1 Introduction
2 Materials
2.1 Human Cell Culture and Bacterial Infection
2.2 Immuno-fluorescence Staining
3 Methods
3.1 Seeding of HeLa Cells
3.2 Infection with Salmonella
3.3 Immuno-fluorescence Staining
3.4 Laser Scanning Confocal Microscopy and Image Processing
4 Notes
References
Chapter 15: Detection and Analysis of Cell Cycle-Associated APC/C-Mediated Cellular Ubiquitylation In Vitro and In Vivo
1 Introduction
2 Materials
2.1 In Vitro Analysis Methods of APC/C-­Mediated Ubiquitylation
2.2 In Vivo Analysis Methods of APC/C-­Mediated Ubiquitylation
3 Methods
3.1 General Methods
3.1.1 In Vitro Production of Unlabeled or [35S]-Methionine-­Labeled Proteins
3.1.2 Cell Cycle Synchronization Protocols
Nocodazole Block
Double-Thymidine Block
Thymidine-­Nocodazole Block
3.1.3 Cells Extracts Preparation
3.2 APC/C-Catalyzed In Vitro Substrate Ubiquitylation
3.3 APC/C-Triggered In Vitro Substrate Degradation
3.4 APC/C-Instigated In Vivo Substrate Ubiquitylation
3.5 APC/C-Mediated In Vivo Substrate Degradation
3.5.1 Downregulation and Overexpression of APC/C Co-activators
Cdh1
Cdc20
3.5.2 Downregulation of APC/C Subunits
3.5.3 Biochemical Inhibition of the APC/C
3.6 Binding Assays Using APC/C Subunits
3.7 Regulation of APC/C Subunits by Posttranslational Modifications
3.7.1 In Vitro Kinase Assays
4 Notes
References
Chapter 16: Detection and Analysis of SUMOylation Substrates In Vitro and In Vivo
1 Introduction
2 Materials
2.1 Materials for In Vitro Experiments Aimed to Detect SUMOylation of Recombinantly Purified Substrates
2.2 Materials for Semi-In Vivo Experiments Aimed to Detect SUMOylation of Substrates Produced in Rabbit Reticulocytes Extracts
2.3 Materials for In Vivo Experiments Aimed to Detect SUMOylated Proteins in Cells
2.4 Materials for Semi-In Vivo Experiments Aimed to Detect SUMOylated Proteins in E. coli
3 Methods
3.1 In Vitro Experiments Aimed to Detect SUMOylated Substrates
3.2 Semi-In Vivo Experiments Aimed to Detect SUMOylated Proteins Using Rabbit Reticulocyte Extracts
3.2.1 Production of the Radiolabeled Substrate for SUMOylation
3.2.2 SUMOylation Assay Using the Radiolabeled Substrate
3.3 Methodology for In Vivo Experiments Aimed to Detect SUMOylated Substrates
3.3.1 Optional Protocol
3.4 Methodology for Semi-In Vivo Experiments Aimed to Detect SUMOylated Proteins in E. coli
4 Notes
References
Chapter 17: Detection of Protein–Protein Interactions and Posttranslational Modifications Using the Proximity Ligation Assay: Application to the Study of the SUMO Pathway
1 Introduction
2 Material
2.1 PLA Kit
3 Methods
3.1 Preparation of Cells
3.1.1 Adherent Cells
3.1.2 Suspension Cells
3.2 Proximity Ligation Assay
3.3 Imaging and Image Analysis
3.4 Application of PLA to the Study of SUMOylation
3.4.1 Detection of Protein SUMOylation
3.4.2 SUMOylated c-Fos Is Found in Actively Transcribed Chromatin
3.4.3 Localization of SUMOylated Proteins on Active of Repressed Chromatin
4 Notes
References
Chapter 18: Dissecting SUMO Dynamics by Mass Spectrometry
1 Introduction
2 Materials
2.1 Lysate Preparation and SUMO Conjugate Purification
2.2 FASP
2.3 Equipment
2.4 Data Analysis
3 Methods
3.1 Test Dynamics of SUMO Modification Changes
3.2 Lysate Preparation
3.3 IMAC Purification
3.4 Antibody Affinity Purification (See Notes 1–4 and 12)
3.5 Filter Aided Sample Prep (FASP)
3.6 Mass Spectrometry
3.7 Data Analysis
4 Notes
References
Chapter 19: Isolation of Lysosomes from Mammalian Tissues and Cultured Cells
1 Introduction
2 Materials
2.1 Isolation of Lysosomes from  Rat Liver
2.2 Isolation of Lysosomes from Cultured Cells
3 Methods
3.1 Isolation of Lysosomes from Rat Liver (Adapted from Wattiaux et al. [15])
3.2 Isolation of Lysosomes from Cultured Cells (Adapted from Storrie and Madden [16])
4 Notes
References
Chapter 20: Analysis of Relevant Parameters for Autophagic Flux Using HeLa Cells Expressing EGFP-LC3
1 Introduction
2 Materials
2.1 Reagents
2.2 Other Materials and Equipment
3 Methods
3.1 DNA Transfection
3.2 siRNA Reverse Transfection
3.3 Microscopy Analysis of GFP-LC3 Fluorescence
3.4 Western Blot Analysis
4 Notes
References
Chapter 21: Analysis of Protein Oligomeric Species by Sucrose Gradients
1 Introduction
2 Materials
2.1 Sucrose Gradient Solutions
3 Methods
3.1 Preparation of Yeast Cells
3.2 Preparation of Mammalian Cells
3.3 Sucrose Gradients
4 Notes
References
Chapter 22: Analysis of Protein Oligomerization by Electrophoresis
1 Introduction
2 Materials
2.1 Cell Lysis
2.2 Electrophoresis and Immunoblotting
2.3 Cross-Linking Reagents
3 Methods
3.1 Casting the Gel
3.2 Sample Preparation and Protein Loading
3.3 Electrophoresis Conditions
3.4 Transfer Conditions
4 Notes
References
Chapter 23: Blot-MS of Carbonylated Proteins: A Tool to Identify Oxidized Proteins
1 Introduction
2 Reagents and Solutions
2.1 Mitochondria Isolation
2.2 Determination of Protein Concentration
2.3 Sodium Dodecyl Sulfate Electrophoresis
2.4 Isoelectric Focusing Electrophoresis and Two-­Dimensional Gel Electrophoresis
2.5 Sample Preparation for IEF and Carbonylated Protein Detection
2.6 Immunochemical Detection
2.7 Tryptic Digestion and LC–MS
3 Methods
3.1 Mitochondria Enrichment
3.2 IEF of Mitochondria Extracts
3.3 Strip Preparation for Protein Carbonyls Detection
3.4 Two-Dimensional Gel Electrophoresis
3.5 Immunochemical Detection
3.6 Image Acquisition and Analysis
3.7 Protein Digestion
3.8 LC Separation and Mass Spectrometry
3.9 Peptide Separation
3.10 MALDI Spectra Acquisition
3.11 Protein Identification and  PTM Analysis
3.12 Data Analysis Using the Bioinformatic Tools: ClueGO and CluePedia
4 Notes
References
Chapter 24: Quantitation of Protein Translation Rate In Vivo with Bioorthogonal Click-Chemistry
1 Introduction
2 Materials
2.1 Cell Culture Media
2.2 Protein Extraction and Quantification
2.3 Click-It Reaction
2.4 Purification of Biotinylated Proteins
2.5 SDS Polyacrylamide Gel Electrophoresis (SDS-PAGE)
2.6 Protein Transfer from SDS-PAGE Gel to Membrane
2.7 Protein Immunodetection by Western Analysis
2.8 Estimation of Protein Synthesis Rate
3 Methods
3.1 Amount of Starting Biological Material
3.2 Total Protein Extraction and Quantification
3.3 Performing the Click-It Reactions
3.4 Purification of Biotinylated “De Novo” Synthesized Proteins
3.5 Separation of the Purified Biotinylated Proteins in a SDS-PAGE
3.6 Protein Transfer from SDS-PAGE Gel to Membrane
3.7 Protein Immunodetection by Western Analysis
3.8 Estimation of Protein Synthesis Rate
4 Notes
References
Chapter 25: A Simple Protocol for High Efficiency Protein Isolation After RNA Isolation from Mouse Thyroid and Other Very Small Tissue Samples
1 Introduction
2 Materials
2.1 Required Chemicals, Commercial Reagents, Kits, and Equipment
2.2 Solutions to be Prepared Before Starting the Procedure
3 Methods
3.1 RNA Isolation
3.2 Protein Isolation
4 Notes
References
Chapter 26: Monitoring Target Engagement of Deubiquitylating Enzymes Using Activity Probes: Past, Present, and Future
1 Ubiquitin–Proteasome System
2 DUBs
3 Activity Probes
4 Activity Probes for Monitoring DUB Activity in Cells
5 Activity Probes for Monitoring DUB Activity in Tissues, Viruses, or Parasites
6 Chemical Proteomics-Activity Probes for Characterizing DUB Inhibitors
7 Activity Probes for Ubiquitin-Like Deconjugating Enzymes
8 Activity Probes Using Ubiquitin Chains or Modified Ubiquitin
9 Activity Probes to Measure Target Engagement
10 What Is Next for Chemical Probes Targeting DUBs?
11 Summary
References
Chapter 27: Activity Based Profiling of Deubiquitylating Enzymes and Inhibitors in Animal Tissues
1 Introduction
2 Materials
2.1 Tissue Homogenization Components
2.2 Protein Quantitation and Activity Probe Assay Components
2.3 Immunoblotting Components
3 Methods
3.1 Animal Welfare and Procedures
3.2 Tissue Homogenization
3.3 Quantitation of Lysates and Incubation with Activity Probe
3.4 SDS-PAGE and Western Blotting
4 Notes
References
Chapter 28: High-Throughput siRNA Screening Applied to the Ubiquitin–Proteasome System
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Chemical Inhibitors
2.3 siRNA Transfection
2.4 siRNA
2.5 Nuclear Counter Stain
2.6 Plates
2.7 Immuno
2.8 Liquid Handling
2.9 Automated Fluorescence Microscopy
2.10 Software
2.11 Statistical Analysis
3 Methods
3.1 Cell Culture
3.2 MG132 or Bortezomib Treatment
3.3 Cycloheximide Treatment
3.4 Library Dilution and Storage
3.5 Standard Screening Protocol for 384-­Well Plates
3.6 Liquid Handling
3.7 Image Acquisition
3.8 Image Analysis
3.9 Statistical Analysis
4 Notes
References
Chapter 29: High-Throughput Yeast-Based Reporter Assay to Identify Compounds with Anti-inflammatory Potential
1 Introduction
2 Materials
2.1 Saccharomyces cerevisiae Strains
2.2 Growth and Storage Media
2.3 Reagents for Monitoring β-Galactosidade Activity Using Ortho-­nitrophenyl-­β-­galactoside
2.4 Reagents for Monitoring β-Galactosidade Activity in Solid Medium
2.5 Equipment Required
3 Methods
3.1 Yeast Cultures and Cell Treatments
3.2 High-Throughput Screening of Compounds with Potential Anti-­inflammatory Activity: Monitoring β-Galactosidase Activity Using Ortho-­nitrophenyl-­β-­galactoside
3.3 Validation of the Anti-­inflammatory Potential of Candidate Compounds: Monitoring β-Galactosidase Activity Using 5-Bromo-4-­chloro-3-indolyl-β-d-­galactopyranoside
4 Notes
References
Chapter 30: Using AlphaScreen® to Identify Small-Molecule Inhibitors Targeting a Conserved Host–Pathogen Interaction
1 Introduction
2 Materials
2.1 Platform
2.2 Environment
2.3 Tools and Reagents
2.4 Storage
3 Methods
3.1 Characterization of the AlphaScreen® Assay as Model for a Conserved Host–Pathogen Interaction
3.1.1 Interaction Assay
3.1.2 Stoichiometry
3.1.3 Sensitivity
3.1.4 Assay Robustness (Z Prime Value)
3.2 Preparation of Intermediate 96-Well and Final 384-Well Prestwick Library Plates
3.3 Screening of the Prestwick Library Using AlphaScreen® Assay
3.4 Data Analysis and Identification of Inhibitors Targeting a Conserved Host-Pathogen Interaction
3.5 AlphaScreen® TruHits Assays
4 Notes
References
Chapter 31: Global MS-Based Proteomics Drug Profiling
1 Introduction
2 Materials
2.1 Subcellular Fractionation
2.2 Organelle Protein Extraction
2.3 Filter-Aided Sample Preparation
2.4 On-Column Stable Isotope Dimethyl Labeling
3 Methods
3.1 Subcellular Fractionation
3.2 Organelle Protein Extraction
3.3 Filter-Aided Sample Preparation (FASP)
3.4 On-Column Stable Isotope Dimethyl Labeling
4 Notes
References
Index