Nucleosomes, Histones and Chromatin Part A - 1st Edition - ISBN: 9780123919403, 9780123919410

Nucleosomes, Histones and Chromatin Part A, Volume 512

1st Edition

Serial Volume Editors: Carl Wu C. Allis
Hardcover ISBN: 9780123919403
eBook ISBN: 9780123919410
Imprint: Academic Press
Published Date: 31st August 2012
Page Count: 390
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Table of Contents

Series Page

Contributors

Preface

Volume in series

Chapter One Examining Histone Posttranslational Modification Patterns by High-Resolution Mass Spectrometry

1 Introduction

2 Materials

3 Nuclei Isolation

4 Acid Extraction

5 Offline Reverse-Phase High-Performance Liquid Chromatography for Histone Variant Purification (Optional)

6 Chemical Derivatization of Histones

7 Stage-Tip Cleanup of Peptide Samples Prior to MS Analysis

8 Enrichment for Phosphorylated Peptides Using Titanium Dioxide (TiO2) Beads (Optional)

9 Online RP-HPLC and MS

Chapter Two Investigating the Cell Cycle-Associated Dynamics of Histone Modifications Using Quantitative Mass Spectrometry

1 Introduction

2 Experimental Design

3 Cell Culture

4 Extraction and Separation of Histones

5 Preparation of Propionic Anhydride-Derivatized Tryptic Histone Peptides

6 Mass Spectrometric Analysis and Quantification of Histone PTMs

Chapter Three A Method to Site-Specifically Incorporate Methyl-Lysine Analogues into Recombinant Proteins

1 Introduction

2 Protocol to Install MLA into Recombinant Proteins

Chapter Four Methods Applied to the Study of Protein Arginine Methylation

1 Introduction

2 In Vitro Arginine-Methylation Assays

3 Enzyme Sources for In Vitro Methylation Reactions

4 Substrate Sources for In Vitro Methylation Assays

5 Cell-Based Methylation Assays

6 Methyl-Specific Antibodies

7 Using Global-Methylation Inhibitors

8 Using Cell-Based Reporter Systems to Screen for PRMT Inhibitors

9 Summary

Chapter Five Enzymatic Analysis of Tet Proteins: Key Enzymes in the Metabolism of DNA Methylation

1 Introduction

2 Expression and Purification of Tet Proteins

3 Tet Activity Assays

4 Analysis of the Cytosine Derivatives by 2D-TLC

5 Analysis of the Cytosine Derivatives by Mass Spectrometry

6 Analysis of the Endogenous Level of Cytosine Derivatives by Mass Spectrometry

Chapter Six Peptide Microarrays to Interrogate the “Histone Code”

1 Introduction

2 Histone Peptide Library

3 Microarray Printing

4 Analysis of Antibodies, Effector Proteins, and Enzymes

5 Data Analysis

6 Summary and Perspectives

Chapter Seven Identifying Chromatin Readers Using a SILAC-Based Histone Peptide Pull-Down Approach

1 Introduction

2 Histone Peptide Synthesis

3 SILAC Labeling

4 Incorporation Check

5 Preparation of Nuclear Extract

6 Histone Peptide Pull-Down

7 In-gel Digestion

8 Mass Spectrometry

9 Data Analysis

10 Outlook

Chapter Eight High-Throughput Strategy to Identify Inhibitors of Histone-Binding Domains

1 Introduction

2 AlphaScreen Assay Principles

3 Materials and Instrumentation

4 Design and Preparation of Histone Peptides and Histidine Tag Fusion Proteins

5 Initial Optimization of an AlphaScreen-Based Histone-Binding Assay

6 Primary Screening

7 Hit Selection and Secondary Screening

8 Concentration-Dependence Validation Studies

9 Assay-Specific Compound Interference

10 Additional Assays for Validation

11 Summary

Chapter Nine Validation of Histone-Binding Partners by Peptide Pull-Downs and Isothermal Titration Calorimetry

1 Introduction

2 Initial Screening: Discovering Putative Interactions Between Histone Peptides and Potential Binding Partners

3 Peptide Pull-downs: Establishing Candidates for Quantitative Assessment

4 Isothermal Titration Calorimetry (ITC): Defining Histone PTM Binding Affinities

5 Reagent Considerations

6 Summary: Peptide Pull-Downs and ITC in the Chromatin Field

Chapter Ten Analysis of Histone Chaperone Antisilencing Function 1 Interactions

1 Introduction

2 Preparation of Asf1

3 Preparation of Histone Components

4 Fluorescence Spectroscopy Analysis of Asf1 Binding Interactions with Histones

5 Electrophoretic Mobility Shift Analysis of Asf1 Binding Interactions with Histones

6 Summary and Implications

Chapter Eleven Quantifying Chromatin-Associated Interactions

1 Introduction to Chromatin and the HI-FI System

2 Fluorescent Labeling and Preparation of Nucleosome Components

3 Imaging Requirements and Reaction Conditions

4 HI-FI Binding Affinity Assay Setup and Execution

5 HI-FI Stoichiometric Assays Setup and Execution

6 HI-FI Competition Assays with FRET

7 Summary

Chapter Twelve Biochemical Assay for Histone H2A.Z Replacement by the Yeast SWR1 Chromatin Remodeling Complex

1 Introduction

2 Preparation of Reagents

3 In Vitro Htz1 Replacement Assay by Chromatin Remodeling Complex SWR1

4 Special Notes

5 Concluding Remarks

Chapter Thirteen Experimental Analysis of the Mechanism of Chromatin Remodeling by RNA Polymerase II

1 Introduction

2 Materials and Methods

3 DNASE I Footprinting of ECs Formed by E. coli RNAP During Transcription Through a Nucleosome

4 Mapping of ECs Formed by Pol II During Transcription Through a Nucleosome Using Restriction Endonucleases

Author Index

Subject Index


Description

Series Page

Contributors

Preface

Volume in series

Chapter One Examining Histone Posttranslational Modification Patterns by High-Resolution Mass Spectrometry

1 Introduction

2 Materials

3 Nuclei Isolation

4 Acid Extraction

5 Offline Reverse-Phase High-Performance Liquid Chromatography for Histone Variant Purification (Optional)

6 Chemical Derivatization of Histones

7 Stage-Tip Cleanup of Peptide Samples Prior to MS Analysis

8 Enrichment for Phosphorylated Peptides Using Titanium Dioxide (TiO2) Beads (Optional)

9 Online RP-HPLC and MS

Chapter Two Investigating the Cell Cycle-Associated Dynamics of Histone Modifications Using Quantitative Mass Spectrometry

1 Introduction

2 Experimental Design

3 Cell Culture

4 Extraction and Separation of Histones

5 Preparation of Propionic Anhydride-Derivatized Tryptic Histone Peptides

6 Mass Spectrometric Analysis and Quantification of Histone PTMs

Chapter Three A Method to Site-Specifically Incorporate Methyl-Lysine Analogues into Recombinant Proteins

1 Introduction

2 Protocol to Install MLA into Recombinant Proteins

Chapter Four Methods Applied to the Study of Protein Arginine Methylation

1 Introduction

2 In Vitro Arginine-Methylation Assays

3 Enzyme Sources for In Vitro Methylation Reactions

4 Substrate Sources for In Vitro Methylation Assays

5 Cell-Based Methylation Assays

6 Methyl-Specific Antibodies

7 Using Global-Methylation Inhibitors

8 Using Cell-Based Reporter Systems to Screen for PRMT Inhibitors

9 Summary

Chapter Five Enzymatic Analysis of Tet Proteins: Key Enzymes in the Metabolism of DNA Methylation

1 Introduction

2 Expression and Purification of Tet Proteins

3 Tet Activity Assays

4 Analysis of the Cytosine Derivatives by 2D-TLC

5 Analysis of the Cytosine Derivatives by Mass Spectrometry

6 Analysis of the Endogenous Level of Cytosine Derivatives by Mass Spectrometry

Chapter Six Peptide Microarrays to Interrogate the “Histone Code”

1 Introduction

2 Histone Peptide Library

3 Microarray Printing

4 Analysis of Antibodies, Effector Proteins, and Enzymes

5 Data Analysis

6 Summary and Perspectives

Chapter Seven Identifying Chromatin Readers Using a SILAC-Based Histone Peptide Pull-Down Approach

1 Introduction

2 Histone Peptide Synthesis

3 SILAC Labeling

4 Incorporation Check

5 Preparation of Nuclear Extract

6 Histone Peptide Pull-Down

7 In-gel Digestion

8 Mass Spectrometry

9 Data Analysis

10 Outlook

Chapter Eight High-Throughput Strategy to Identify Inhibitors of Histone-Binding Domains

1 Introduction

2 AlphaScreen Assay Principles

3 Materials and Instrumentation

4 Design and Preparation of Histone Peptides and Histidine Tag Fusion Proteins

5 Initial Optimization of an AlphaScreen-Based Histone-Binding Assay

6 Primary Screening

7 Hit Selection and Secondary Screening

8 Concentration-Dependence Validation Studies

9 Assay-Specific Compound Interference

10 Additional Assays for Validation

11 Summary

Chapter Nine Validation of Histone-Binding Partners by Peptide Pull-Downs and Isothermal Titration Calorimetry

1 Introduction

2 Initial Screening: Discovering Putative Interactions Between Histone Peptides and Potential Binding Partners

3 Peptide Pull-downs: Establishing Candidates for Quantitative Assessment

4 Isothermal Titration Calorimetry (ITC): Defining Histone PTM Binding Affinities

5 Reagent Considerations

6 Summary: Peptide Pull-Downs and ITC in the Chromatin Field

Chapter Ten Analysis of Histone Chaperone Antisilencing Function 1 Interactions

1 Introduction

2 Preparation of Asf1

3 Preparation of Histone Components

4 Fluorescence Spectroscopy Analysis of Asf1 Binding Interactions with Histones

5 Electrophoretic Mobility Shift Analysis of Asf1 Binding Interactions with Histones

6 Summary and Implications

Chapter Eleven Quantifying Chromatin-Associated Interactions

1 Introduction to Chromatin and the HI-FI System

2 Fluorescent Labeling and Preparation of Nucleosome Components

3 Imaging Requirements and Reaction Conditions

4 HI-FI Binding Affinity Assay Setup and Execution

5 HI-FI Stoichiometric Assays Setup and Execution

6 HI-FI Competition Assays with FRET

7 Summary

Chapter Twelve Biochemical Assay for Histone H2A.Z Replacement by the Yeast SWR1 Chromatin Remodeling Complex

1 Introduction

2 Preparation of Reagents

3 In Vitro Htz1 Replacement Assay by Chromatin Remodeling Complex SWR1

4 Special Notes

5 Concluding Remarks

Chapter Thirteen Experimental Analysis of the Mechanism of Chromatin Remodeling by RNA Polymerase II

1 Introduction

2 Materials and Methods

3 DNASE I Footprinting of ECs Formed by E. coli RNAP During Transcription Through a Nucleosome

4 Mapping of ECs Formed by Pol II During Transcription Through a Nucleosome Using Restriction Endonucleases

Author Index

Subject Index

Key Features

  • Continues the legacy of this premier serial by containing quality chapters authored by leaders in the field
  • The first of 2 volumes covering nucleosomes, histones and chromatin
  • Chapters on methods applied to the study of protein arginine methylation, high-resolution identification of intra- and interchromosomal DNA interactions by 4C technology, and peptide arrays to interrogate the binding specificity of chromatin-binding proteins

Readership

Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists


Details

No. of pages:
390
Language:
English
Copyright:
© Academic Press 2012
Published:
Imprint:
Academic Press
eBook ISBN:
9780123919410
Hardcover ISBN:
9780123919403

About the Serial Volume Editors

Carl Wu Serial Volume Editor

Affiliations and Expertise

National Cancer Institute, Bethesda, MD

C. Allis Serial Volume Editor

Affiliations and Expertise

The Rockefeller University, New York, NY