A Laboratory Guide to In Vitro Studies of Protein-DNA Interactions

Abstract

I Introduction.- The Study of Protein-DNA Interactions by Means of Enzymes.- II The study of Protein-DNA Interactions by Deoxyribonuclease 1 Footprinting.- III Exonuclease III Protection Assay for Specific DNA-Binding Proteins.- IV The Proteolytic Clipping Band-Shift Assay of Protein-DNA Complexes.- The Study of Protein-DNA Interactions by UV Light.- V Visualising Intimate Protein-DNA Contacts and Altered DNA Structures with Ultraviolet Light.- VI UV Cross-Linking of Protein to Bromouridine-Substituted RNA.- VII Molecular Weight Determination of DNA-Binding Proteins by UV Cross-Linking Combined with SDS Polyacrylamide Gel Electrophoresis.- The Study of Protein-DNA Interactions by Means of Chemicals (Interference Assays).- VIII Interaction of Proteins with Partially Depurinated and Depyrimidinated Oligonucleotides (Missing-Contact Probing of Protein-DNA Interactions).- IX The Study of Protein-DNA Contacts by Ethylation Interference.- X The Study of Protein-DNA Interactions by Methylation Interference.- XI Using the Chemistry of the Hydroxyl Radical to Determine Structural Details About DNA and Protein-DNA Complexes.- XII Detection of Unusual DNA Structures and DNA-Protein Interactions by Diethylpyrocarbonate Carbethoxylation.- The Study of Protein-DNA Interactions by Means of Electron Microscopy.- XIII Electron Microscope Visualisation of Protein-DNA Complexes.- The Study of Protein-DNA Interactions by Gel Mobility-shift Assays.- XIV Qualitative and Quantitative Studies of Protein-DNA Interactions by Gel Mobility-Shift Assay.- XV Diagonal Gel Mobility-Shift Assays for the Resolution of Multi-subunit Complexes Binding to Regulatory Elements of Specific Genes.- XVI A Rapid Gel-Shift Technique to Analyze DNA-Protein Interactions Using PhastSystem (TM).- Protein-DNA Interactions on a Solid Support.- XVII Purification of Sequence-Specific DNA-Binding Proteins by Affinity Chromatography.- XVIII Cloning of Sequence-Specific DNA-Binding Proteins by Screening ? cDNA Expression Libraries with Radiolabelled Binding-Site Probes.- The Study of Protein-DNA Interaction Using Special Vectors.- XIX Detection of DNA Bending by Gel Electrophoresis: Use of Plasmid Vectors.- Other Related Techniques.- XX The Use of DNA Amplification for In Vitro Footprinting Experiments.- XXI Non-radioactive Detection Methods for In Vitro Footprinting.- XXII Quantitative Determination of Proteins in Crude Extracts.- Appendices.- Appendix A DNA Isolation from E. coli (J.P. Jost).- Appendix B The Spun Column Procedure.- Appendix C Purification of Oligonucleotides (J. Jiricny).- Appendix D Migration of DNA on Native and Denaturating Gels of Different Polyacrylamide Concentration.- Appendix E Preparation of a Nuclear Lysate from Liver (J.P. Jost).- Appendix F Preparation of a Cell Lysate from Tissue Culture Cells.- Appendix G Preparation of Nuclear Extracts from Cells in Tissue Culture.- Appendix H Inhibitors of Proteases (E. Shaw).- Appendix I The Most Common Protein Kinase and Phosphatase Inhibitors (B. Hemmings).- Appendix J Tables on the Precipitation of Proteins with Ammonium Sulphate.