Chaperones Methods and Protocols 2nd Edition by John M Walker ISBN 9781071633427 1071633414

Chaperones Methods and Protocols 2nd Edition by John M Walker – Ebook PDF Instant Download/Delivery: 9781071633427, 1071633414
Full download Chaperones Methods and Protocols 2nd Edition after payment

Product details:
ISBN 10: 1071633414
ISBN 13: 9781071633427
Author: John M Walker

This second edition volume expands on the previous edition with new discussions on the latest techniques used to study molecular chaperones and the stress response. The chapters in this book cover such as analysis of the initiation and regulation of the stress response; the role of heat shock protein 90 (Hsp90) in gene expression through chromosome-immunoprecipitation; features of chaperone function and biology; the emerging role of the extracellular HSPs; and the use of chaperones as biomarkers. 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.
Cutting-edge and thorough, Chaperones: Methods and Protocols, Second Edition is a valuable resource for all researchers who want to learn more about this interesting and developing field

Chaperones Methods and Protocols 2nd Edition Table of contents:

Chapter 1: Monitoring of the Heat Shock Response with a Real-Time Luciferase Reporter
1 Introduction
2 Materials
2.1 Cell Culture and Transfection
2.2 Plastic Ware
2.3 Plasmids
2.4 Buffers and Reagents
2.5 Equipment
3 Methods
3.1 Cell Plating
3.2 Transfection
3.3 Drug Treatment (Optional)
3.4 Heat Shock
3.5 Cell Lysis and Sample Storage
3.6 Luciferase Assay
3.7 β-Galactosidase Assay
3.8 Normalization and Statistics
4 Results
5 Notes
References
Chapter 2: Studying RNA Polymerase II Promoter-Proximal Pausing by In Vitro Immobilized Template and Transcription Assays
1 Introduction
2 Materials
2.1 Preparation of the HSP70 Template DNA
2.2 Preparation of the HSP70 Immobilized Template
2.3 Performing the Immobilized Template Assay
2.4 Performing In Vitro Transcription Assay
2.5 Running Denaturing Polyacrylamide Gel Electrophoresis
3 Methods
3.1 PCR Amplification of the HSP70 Template DNA
3.2 Conjugating Biotinylated DNA with Streptavidin-Coated Beads
3.3 Immobilized Template Assay
3.4 In Vitro Transcription Assay
3.5 Running Denaturing Polyacrylamide Gel Electrophoresis
4 Notes
References
Chapter 3: Role of Heat Shock Factors in Stress-Induced Transcription: An Update
1 Introduction
2 Materials
2.1 ChIP Assay Buffers and Reagents
3 Methods
3.1 ChIP Assay Buffers and Reagents
4 Notes
References
Chapter 4: A Workflow Guide to RNA-Seq Analysis of Chaperone Function and Beyond
1 Introduction
2 Materials
2.1 Samples
2.2 Tissue Harvest and RNA Isolation
2.3 Assessment of the Concentration, Purity, and Integrity of the RNA Sample
2.4 cDNA Library Construction (Service Often Available at Next-Generation Sequencing Facility)
2.5 Next-Generation Sequencing
2.6 Sequence Processing and Analysis
3 Methods
3.1 Experimental Design
3.2 RNA Collection from Cell Culture
3.3 RNA Extraction from Mouse Tissue
3.4 Assess the Concentration and Integrity of the RNA Sample
3.5 Stranded cDNA Library Construction Using KAPA Stranded RNA-Seq Kit with RiboErase for Illumina Platforms
3.6 Next-Generation Sequencing
3.7 Sequence Processing and Analysis
4 Notes
4.1 Experimental Design
4.2 RNA Collection
4.3 RNA Integrity Analysis
4.4 cDNA Library Construction
4.5 Next-Generation Sequencing
4.6 Sequence Processing and Analysis
4.6.1 Quality Control of Raw Reads
4.6.2 Alignment to the Reference Genome
4.6.3 Differential Expression Analysis
4.6.4 Functional Enrichment Analysis Using clusterProfiler
4.6.5 Representation of Data
4.7 Additional Notes
References
Chapter 5: Chromatin Immunoprecipitation (ChIP) of Heat Shock Protein 90 (Hsp90)
1 Introduction
2 Materials
2.1 Cells Preparation and Cross-Linking
2.2 Chromatin Extraction and Sonication
2.3 Chromatin Quality Check
2.4 Immunoprecipitation
2.4.1 Equipment
3 Methods
3.1 Cells Preparation and Cross-Linking
3.2 Chromatin Extraction and Sonication
3.3 Chromatin Quality Check
3.4 Immunoprecipitation
4 Notes
References
Chapter 6: Transfection and Thermotolerance Methods for Analysis of miR-570 Targeting the HSP Chaperone Network
1 Introduction
2 Materials
2.1 Transfection
2.2 Heat Shock/Thermotolerance Conditions
2.3 Protein Assay
2.3.1 Sample Preparation for Western Blotting
2.3.2 SDS-PAGE
2.3.3 Protein Transfer
2.3.4 Immunoblotting
2.3.5 Imaging and Data Analysis
2.4 Cell Proliferation Assay
2.5 Colony Formation Assay
3 Methods
3.1 Transfection
3.2 Heat Shock/Thermotolerance Conditions
3.3 Protein Assay
3.3.1 RIPA Buffer and Homogenization Protocol
3.3.2 Sample Preparation for SDS-PAGE
3.3.3 SDS-PAGE
3.3.4 Wet Transfer
3.3.5 Immunoblotting
3.4 Cell Proliferation Assay
3.5 Colony Formation Assay
4 Notes
References
Chapter 7: Targeted Replacement of HSF1 Phosphorylation Sites at S303/S307 with Alanine Residues in Mice Increases Cell Prolif…
1 Introduction
2 Materials
2.1 Reagents
2.1.1 Molecular Biology
2.1.2 Southern Blot
2.1.3 Cell Culture and Histology
2.1.4 Drug for Selection of Clones and Treatment
2.2 Enzymes
2.3 Plasmids
2.4 Commercial Kits
3 Methods
4 Physiological Effects of HSF1 Following S303/S307 Mutations to Alanine Residues
4.1 Loss of HSF1 Phosphorylation at S303/S307 Promotes Cell Proliferation, Drug Resistance, and Tumorigenesis
5 Conclusions
References
Chapter 8: Bimolecular Fluorescence Complementation Assay to Evaluate HSP90-Client Protein Interactions in Cells
1 Introduction
2 Materials
2.1 Equipment
3 Methods
4 Notes
References
Chapter 9: Complementation Assays for Co-chaperone Function
1 Introduction
2 Materials
2.1 Prokaryotic Complementation Assay Materials
2.2 Eukaryotic Complementation Assay Materials
3 Methods
3.1 Prokaryotic Complementation Assay Protocol
3.2 Eukaryotic Complementation Assay Protocol
4 Notes
References
Chapter 10: Optimized Microscale Protein Aggregation Suppression Assay: A Method for Evaluating the Holdase Activity of Chaper…
1 Introduction
2 Materials
2.1 Plasmids
2.2 Buffers and Stock Solutions
3 Methods
3.1 His-EcDnaK Protein Purification
3.2 Kinetic and Endpoint Analysis of Aggregation Suppression Analysis Using Absorbance at 360 nm
3.3 Kinetic and Endpoint Analysis of Aggregation Suppression Analysis Fluorescence-Based Detection
3.4 Data Analysis
4 Notes
References
Chapter 11: Detecting Posttranslational Modifications of Hsp90 Isoforms
1 Introduction
2 Materials
3 Methods
3.1 Extraction of Total Yeast Protein
3.2 Extraction of Total Protein from HEK293 Cells and Immunoprecipitation (IP) of hHsp90
3.3 Mitochondrial Isolation from HEK293 Cells, Disruption, and IP of TRAP1
3.4 Western Blotting and Detection of Hsp90 PTMs
4 Notes
References
Chapter 12: Multiple Targeting of HSP Isoforms to Challenge Isoform Specificity and Compensatory Expression
1 Introduction
2 Materials
2.1 Transfection
2.1.1 Cell Culture for Transfection
2.1.2 Preparation of siRNAs for Transfections
2.2 Simple Isolation of Exosomes/EVs
2.2.1 Cell Culture for Exosome/EV Isolation
2.2.2 Isolation of Exosomes/EVs Using the PBP Method
2.3 Buffers to Lyse Membranes of Cells and Vesicles
2.4 Protein Assay
2.5 Sample Preparation for Western Blotting
2.6 SDS-Page
2.7 Protein Transfer
2.8 Immunoblotting
2.9 Imaging and Data Analysis
3 Methods
3.1 Transfection
3.1.1 Cell Culture and Transfection
3.2 Cell Culture and Isolation of EVs/Exosomes Using the PBP Method
3.2.1 2D Cell Culture for Preparation of sEV/Exosomes and WCL
3.2.2 (Optional) Removal of Large EVs
3.2.3 Concentrating the EV Fraction
3.2.4 Isolation of EVs
3.2.5 Non-vesicular Fraction (Including Vesicle-Free HSP90 Proteins)
3.3 Harvest of Cellular Proteins (WCL)
3.3.1 Common Steps
3.3.2 Cell Lysis Buffer Protocol
3.3.3 RIPA Buffer and Homogenization Protocol
3.3.4 Trypsin and RIPA Buffer Protocol
3.3.5 Sample Buffer Protocol
3.4 Protein Assay
3.5 Sample Preparation for Western Blotting
3.6 SDS-Page
3.7 Wet Transfer
3.8 Immunoblotting
3.9 Stripping
4 Notes
References
Chapter 13: Using a Modified Proximity Ligation Protocol to Study the Interaction Between Chaperones and Associated Proteins
1 Introduction
1.1 Results
1.1.1 Using the Modified PLA Method for Detection of Complexes of Hsp70-Bag3 with Components of the Hippo Pathway
1.2 Modified Proximity Ligation IPAD Technology
2 Materials
3 Methods
3.1 Conjugation Between Antibodies and Oligonucleotides
3.2 Antibody Purification
3.3 IPAD Protocol
4 Notes
References
Chapter 14: Use of Native-PAGE for the Identification of Epichaperomes in Cell Lines
1 Introduction
2 Materials
2.1 Cell Culture
2.2 Cell Lysate Preparation for Native-PAGE
2.3 Native Polyacrylamide Gel
2.4 Membrane Stripping
2.5 SDS-Page
2.6 Detection
3 Methods
3.1 Cell Culture and Cell Lysate Preparation for Native- and SDS-PAGE
3.2 Cell Lysate Preparation for SDS-PAGE
3.3 Preparation of Precast Gels for Running Native-PAGE
3.4 Preparation of Handmade Continuous Gels for Running Native-PAGE
3.5 SDS-Page
3.6 Protein Transfer and Immunoblotting
3.7 Chemiluminescent Detection
4 Notes
References
Chapter 15: Molecular Chaperone Receptors: An Update
1 Introduction
2 Materials
3 Methods
3.1 Screening for HSP Receptors
3.2 Alexa Fluor 488-Labeled Purified HSP70 Preparation
3.3 Alexa Fluor 488-Labeled Purified Hsp90 Preparation
3.4 Alexa Fluor 488-Labeled Purified DnaK Preparation
3.5 HSP Binding Assay
3.6 Studying HSP-SREC-I Interaction In Vivo
3.7 shRNA Directed Against SREC-I
4 Notes
References
Chapter 16: A Novel Heat Shock Protein 70-Based Vaccine Prepared from DC Tumor Fusion Cells: An Update
1 Introduction
2 Materials
2.1 Isolation of Tumor Cells from Patient-Derived Solid Sample or Malignant Fluid
2.2 Generation of DC from Human Peripheral Blood Monocytes
2.3 Preparation of DC Tumor Fusions
2.4 Preparation of Hsp70.PC Extraction from DC Tumor Fusions
2.5 Measurement of Levels of Endotoxin
3 Methods
3.1 Generation of DC from Human Peripheral Blood Monocytes
3.2 Preparation of Tumor Cells
3.3 Cell Fusion
3.4 Extraction of Hsp70 Peptide Complexes (Hsp70.PC) from DC Tumor Fusion Cell Products
4 Notes
4.1 Cell Fusion
4.2 Extraction of Hsp70 Peptide Complexes
References
Chapter 17: Methods to Assess the Impact of Hsp90 Chaperone Function on Extracellular Client MMP2 Activity
1 Introduction
2 Materials
2.1 Hsp90α:MMP2 Complex Formation
2.2 Immunoblotting (Bio-Rad Criterion System)
2.3 Gelatin Zymography (ThermoFisher Scientific Protein Gel Electrophoresis Chambers, Empty Gel Cassettes, and Buffers)
2.4 MMP2 Fluorometric Enzyme Activity Assay
2.5 Statistic Software
3 Methods
3.1 In Vitro Hsp90α:Active MMP2 Complex Formation
3.2 Protein Detection: Immunoblotting
3.3 MMP2 Gelatinolytic Activity: Gelatin Zymography
3.4 MMP2 Fluorometric Enzyme Activity Assay
References
Chapter 18: Proteomic Profiling of the Extracellular Vesicle Chaperone in Cancer
1 Introduction
2 Materials
2.1 Cell and Tissue Culture
2.1.1 Common Materials
2.1.2 2D or 3D Culture
2.1.3 Tissue-Exudative Extracellular Vesicles (Te-EVs)
2.2 UF Method
2.3 PBP Method
2.4 UC Method
2.5 AP Method (see Notes 10 and 11)
2.6 SEC Method
2.7 Basic EV Analyses
2.8 Proteome Analysis (LC-MS/MS)
3 Methods
3.1 Tissue/Cell Culture
3.1.1 2D Cell Culture
3.1.2 Spheroid Culture and Tumoroid Culture
3.1.3 Te-EVs
3.2 UF Method (See Note 24)
3.3 PBP Method
3.4 UC Method
3.5 AP Method
3.6 SEC Method
3.6.1 Concentration Step
3.6.2 SEC Step
3.6.3 Gathering Fraction Step
3.7 Non-EV Fraction
3.8 Basic EV Analyses
3.8.1 Measure the Numbers and Size Distribution of Vesicles/Particles (Video Drop, qNano, or NanoSight). Alternatively, Measur…
3.8.2 Visualize the Vesicles by Negative Staining and TEM
3.8.3 Measure the Protein Concentration Using a Micro BCA Protein Assay
3.9 Proteome Analysis (LC-MS/MS)
4 Notes
References
Chapter 19: A Modified Differential Centrifugation Protocol for Isolation and Quantitation of Extracellular Heat Shock Protein…
1 Introduction
2 Materials
2.1 For Cell Culture
2.2 For Centrifugations
2.3 For Immunol (Western) Blotting Analysis
3 Methods
3.1 Cell Culture
3.2 Separation of Cell-Conditioned Medium into Three Fractions
3.3 Immunoblotting Analysis
4 Notes
References
Chapter 20: Immunohistochemistry of Human Hsp60 in Health and Disease: Recent Advances in Immunomorphology and Methods for Ass…
1 Introduction
2 Immunomorphological Assessment of Hsp60
2.1 Materials
2.2 Methods
2.2.1 Immunohistochemistry
2.2.2 Immunofluorescence and Confocal Microscopy
3 Assessment of Hsp60 and microRNAs in EVs from Plasma
3.1 Materials
3.1.1 Sample Collection
3.1.2 EV Isolation
3.1.3 EV Morphological Characterization
3.1.4 Protein Isolation and Assessment of Hsp60
3.1.5 microRNA Isolation and Hsp60-Related microRNA Analysis
3.1.5.1 RNA Extraction
3.1.5.2 microRNA Analysis
3.2 Methods
3.2.1 Sample Collection
3.2.2 EV Isolation
3.2.3 EV Morphological Characterization by Transmission Electron Microscopy (TEM)
3.2.4 Protein Isolation and Assessment of Hsp60
3.2.5 microRNA Isolation and Hsp60-Related microRNA Analysis
3.2.5.1 RNA Extraction
3.2.5.2 microRNA Analysis
4 Notes
References
Chapter 21: Multiplex Immunostaining Method to Distinguish HSP Isoforms in Cancer Tissue Specimens
1 Introduction
2 Materials
2.1 Sample Preparation
2.2 Deparaffinization/Rehydration
2.3 Antigen Retrieval
2.4 Immunoreaction
2.5 Immunostaining Components
3 Methods
3.1 Sample Preparation
3.2 Deparaffinization/Rehydration
3.3 Antigen Retrieval
3.4 Immunoreaction (First Cycle)
3.5 Immunostaining (First Cycle: Brown Color)
3.6 Detachment of First Cycle Antibodies from Specimen
3.7 Immunoreaction (Second Cycle)
3.8 Immunostaining (Second Cycle: Red Color)
3.9 Dehydration and Mounting
4 Notes
References
Chapter 22: Large-Scale Databases and Portals on Cancer Genome to Analyze Chaperone Genes Correlated to Patient Prognosis
1 Introduction
2 Materials
3 Methods
3.1 HPA
3.2 KM Plotter
3.3 GEPIA 2
4 Notes
References
Chapter 23: Immunohistochemical, Flow Cytometric, and ELISA-Based Analyses of Intracellular, Membrane-Expressed, and Extracell…
1 Introduction
2 Materials
2.1 Patient-Derived Glioblastoma Multiforme Cell Lines
2.2 Patient- and Preclinical Model-Derived Paraffin-Embedded Tissue
2.3 cmHsp70.1 Anti-Hsp70 Monoclonal Antibody (mAb)
2.4 Immunohistochemistry (IHC) for Detecting Tissue Expression of Hsp70
2.4.1 Deparaffinization Reagents
2.4.2 Target Retrieval and Staining Reagents
2.5 Flow Cytometry for Detecting Cell Surface Expression of Membrane Hsp70
2.5.1 PBS, pH 7.2, Supplemented with Sodium Azide (NaN3) and Fetal Bovine Serum (FBS)
2.6 Enzyme-Linked Immunosorbent Assays (compHsp70 ELISA) for Detecting Extracellular Free and Exosomal Hsp70 (See Fig. 1)
3 Methods
3.1 Immunohistochemistry (IHC) for Detecting Tissue Expression of Hsp70
3.1.1 Rehydration
3.1.2 Target Retrieval and Staining
3.1.3 Dehydration and Embedding
3.1.4 Scoring Criteria of IHC Using cmHsp70.1 mAb
3.2 Flow Cytometry for Detecting Cell Surface Expression of Membrane Hsp70
3.3 Enzyme-Linked Immunosorbent Assays (compHsp70 ELISA) for Detecting Extracellular Free and Exosomal Hsp70
4 Notes
4.1 Immunohistochemistry (IHC) Scoring
4.2 Flow Cytometry for Detecting Cell Surface Expression of Membrane Hsp70
4.3 Enzyme-Linked Immunosorbent Assays (compHsp70 ELISA) for Detecting Extracellular Free and Exosomal Hsp70

People also search for Chaperones Methods and Protocols 2nd Edition:

chaperones and chaperonins
    
chaperonin and chaperones
    
what are chaperone proteins and what do they do
    
what are chaperone proteins and what is their function
    
chaperone protocol

Tags: John M Walker, Chaperones, Protocols, Methods