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Mapping Protein/DNA Interactions by Cross-Linking
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Mapping Protein/DNA Interactions by Cross-Linking
Institut National de la Santé et de la Recherche Médicale
2001
© 2001
Institut National de la Santé et de la Recherche Médicale (INSERM)
molecular biology
Table of Contents
Dissecting Fission Yeast Centromeres via Silencing
Robin Allshire
Introduction
Our Observations
Our Current View
References
Localizing Replication Sites and Nuclear Proteins
Angela Taddei, Danièle Roche, Jean-Baptiste Sibarita, Sébastien Huart, Christèle Maison, Delphine Bailly, and Geneviève Almouzni
Practical Approach
Selected References
Finding Downstream Target DNAs for Chromatin Proteins by X-CHIP in Drosophila
Nicolas Nègre and Giacomo Cavalli
Section I. General Discussion of the Approach
Section II. Detailed Protocol for X-ChIP
References
Emerging Techniques: UV Laser Footprinting and Protein--DNA Crosslinking
Stefan Dimitrov
Laser Protein--DNA Photofootprinting
UV Laser-induced Protein--DNA Crosslinking
Conclusions and Perspectives
References
Nuclear Organization and Gene Expression: Visualization of Transcription and Higher Order Chromatin Structure
Roel van Driel and Pernette J Verschure
Scientific Context
Technical Strategies
Protocol for Simultaneous Labelling of DNA and Antigens in the Cell Nucleus
Literature Cited
Examining the Distribution of Telomeric and DNA Repair Proteins by ChrIP and Real-Time PCR
Karine Dubrana
General Principle of Chromatin ImmunoPrecipitation (ChrIP)
Important Steps to Control
Investing in the Real-Time Technique
Appendix: Chromatin-IP (ChrIP) Protocol
References
Nuclear Organisation and Gene Silencing in Lymphocytes
Amanda Fisher and Matthias Merkenschlager
Direct Visualization of Transcription Factor Binding to Regulatory Elements in Living Cells
Gordon L Hager
Introduction
Tagging Nuclear Receptors with Fluorescent Proteins
Characterization of the Fusion Chimera
Mode of Expression
Instrumentation
Gene Targets
Dynamic Movement of Receptors on Gene Targets
Reference List
Part I. Chromatin Protein Complexes: Dynamic Assemblies in the Nucleus
Donna Jovin
Nuclear Architecture and Functional Consequences
Polycomb-Group Protein Repression Complexes, Cellular Memory
The Drosophila X Chromosome and the MSL Dosage Compensation Complex
Photophysical Methods to Extract Information about Molecular Proximity, Interactions, and Mobilities
References
Isolation of Homeodomain Engrailed Target Genes by UV Cross-Linking and Chromatin IP
Florence Maschat
Part 1: General Discussion of the Research and of the Method
IMAGE
Part 2: A Detailed Method for in Vivo UV Cross-Linking and Chromatin IP
References
Analysis of Chromatin Assembly, Chromatin Domains, and DNA Replication Using Xenopus Systems
Jean-Marc Lemaitre, Yegor Vassetzky, and Marcel Méchali
Collection of Eggs
Low-Speed Egg Extract (LSE)
Preparation of High-Speed Extract (HSE)
Preparation of Demembranated Sperm Nuclei
Assays for DNA Replication in Xenopus Extracts
DNA Replication Assay by TCA Precipitation
Calculation of the Activity of the Extract
Detection of Protein Interaction with Chromatin by Immunolocalisation
Detection of Protein Interaction with Chromatin by a Purification Assay
Protocol for Single-stranded DNA Replication and Chromatin Assembly
Notes
Chromatin Assembly
Supercoiling Assay (First Method)
Supercoiling Assay (Second Method)
Micrococcal Nuclease Digestion Assay
Notes
Studying DNA--Protein Interactions in Vivo Using LM-PCR Coupled with Nuclear Matrix Isolation
Protocol for the Nuclear Matrix Isolation
Ligation-mediated PCR
Appendix: Buffers and Solutions
References
Binding Sights in Chromatin by X-ChIP
Achim Breiling and Valerio Orlando
Formaldehyde Fixation, Chromatin Purification, and Immunoprecipitation for Drosophila SL2 Tissue Culture Cells (Applicable Also to Mouse and Human Cell Lines)
References
Chromatin Immunoprecipitation in Budding Yeast
Tomoyuki Tanaka
Experimental Protocol
References
ChIP with Native Chromatin: Advantages and Problems Relative to Methods Using Cross-Linked Material
Bryan Turner
Part 1. General Discussion
References
Highly Sensitive FISH in Combination with the Visualization of the Mammalian Genome: Architecture
Daniele Zink
Introduction
Results
Discussion
Materials and Methods
References
Part II. Fly-FISHing: A Protocol to Localize Single Copy Genes inside the Nucleus in Whole Mount Embryos
Mark J Gemkow
Introduction
Protocol and Materials
Considerations
Acknowledgments
References
Part 2. Detailed Method for Immunoprecipitation of Unfixed Chromatin from Mammalian Cells
Laura O'Neill and Bryan Turner
A. Chromatin Preparation from Cultured Human Cells
B. Analysis of Chromatin
C. Immunoprecipitation of Unfixed Chromatin (with Antibodies to Acetylated H4)
D. DNA Isolation from Chromatin Fractions
E. Protein Isolation from Chromatin Fractions
F. Solutions
Copyright © 2001
Institut National de la Santé et de la Recherche Médicale (INSERM)
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