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Nucleosomes, Histones and Chromatin Part A
- 1st Edition, Volume 512 - August 31, 2012
- Editors: Carl Wu, C. David Allis
- Language: English
- Hardback ISBN:9 7 8 - 0 - 1 2 - 3 9 1 9 4 0 - 3
- eBook ISBN:9 7 8 - 0 - 1 2 - 3 9 1 9 4 1 - 0
This new volume of Methods in Enzymology continues the legacy of this premier serial by containing quality chapters authored by leaders in the field. The first of 2 volumes coveri… Read more
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Request a sales quoteThis new volume of Methods in Enzymology 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, it has 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.
- 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
Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists
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
- No. of pages: 390
- Language: English
- Edition: 1
- Volume: 512
- Published: August 31, 2012
- Imprint: Academic Press
- Hardback ISBN: 9780123919403
- eBook ISBN: 9780123919410
CW
Carl Wu
Affiliations and expertise
National Cancer Institute, Bethesda, MDCA
C. David Allis
Affiliations and expertise
The Rockefeller University, New York, NYRead Nucleosomes, Histones and Chromatin Part A on ScienceDirect