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This detailed volume explores poly(ADP-ribose) polymerases (PARPs) in the biology of eukaryotes and their relevance to human health. Beginning with a section on the detection and quantification of poly(ADP-ribose) polymer (pADPr), the book continues by delving into the identification of protein targets, functional analysis, the poly(ADP-ribosyl)ating pathway in chromatin and genes expression, as well as the use of animal models and PARP1 inhibitor design and testing, and more. Written for the highly successful Methods in Molecular Biology series, chapters include introductions to their respective topics, lists of the necessary materials and reagents, step-by-step and readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls.
Authoritative and up-to-date, Poly(ADP-Ribose) Polymerase: Methods and Protocols, Third Edition presents essential new and classical methods for studying the pADPr-pathway.
List of contents
Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High Performance Liquid Chromatography, and Capillary Electrophoresis.- Detecting and Quantifying pADPr In Vivo.- Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage.- Analyzing PARP1 Activity: Small Molecule Reactants and Attached Chains of Poly(ADP-Ribose).- Detecting Poly(ADP-Ribose) In Vitro and in Cells Using PAR-Trackers.- An Enzyme-Linked Immunosorbent Assay to Quantify Poly(ADP-Ribose) Level In Vivo.- Subcellular Quantitation of ADP-Ribosylation by High Content Microscopy.- A Simple Method to Study ADP-Ribosylation Reversal: From Function to Drug Discovery.- Immunoprecipitation Using Mono-ADP-Ribosylation Specific Antibodies.- A Clickable NAD+ Analog-Based Assay of Poly(ADP-Ribosyl)ated Proteins.- Functional Analysis of Histone ADP-Ribosylation In Vitro and in Cells.- Studying the Immunomodulatory Functions of PARP1 and PARP2 in Mouse Models of Cancer.- Methods for Investigating Transient Receptor Potential Melastatin-2 (TRPM2), a Cation Channel Activated by ADP-Ribose and Involved in Cell Death.- Methods to Assess the Role of PARPs in Regulating Mitochondrial Oxidative Function.- Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry.- Cytological Approaches to Visualize Intracellular Dynamics of RNA Binding Proteins at Active Genes in Drosophila.- Chromatin Immunoprecipitation Approach to Determine How PARP1 Domains Affect Binding Pattern to Chromatin.- Approach to Measuring the Effect of PARP1 on RNA Polymerase II Elongation Rates.- Examining the Effect of PARP-1 Inhibitors on Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells.- Using Drosophila Genetics to Identify Factors that Affect PARP1 Activity In Vivo.- Generating PARP Knockout D. Melanogaster with CRISPR/Cas9 System.- TaqMan Multiplex qPCR Method to Genotype PARG Knockout Mice.-Cell-Based Screening for New PARP Inhibitors Utilizing PARG Mutated Mouse Embryonic Stem Cells.- Quantification of PARP7 Protein Levels and PARP7 Inhibitor Target Engagement in Cells Using a Split Nanoluciferase System.- Purification of Recombinant Human PARG and Activity Assays.- Purification of Recombinant Human PARP-3.
