Poly(ADP-Ribose) Polymerase: Methods and Protocols

Description

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. Preface Contents Contributors Part I: Detection and Quantification of pADPr Chapter 1: Fluorescence-Based Analyses of Poly(ADP-Ribose) Length by Gel Electrophoresis, High-Performance Liquid Chromatograp... 1 Introduction 2 Materials 2.1 PAR Production 2.2 Cy5 Labeling of PAR by ELTA 2.3 PAR Profile Method 1: HPLC with DAD or FLD Detectors 2.4 PAR Profile Method 2: PAGE with Fluorescent Gel Scanner 2.5 PAR Profile Method 3: CE with LEDIF Detector 3 Methods 3.1 PAR Production 3.1.1 hTNKS1 Expression and Purification 3.1.2 Bulk PAR Preparation 3.1.3 Making Defined Length PAR 3.2 Cy5 Labeling of PAR by ELTA 3.3 PAR Profile Method 1: HPLC with DAD or FLD Detectors 3.4 PAR Profile Method 2: PAGE with Fluorescent Gel Scanner 3.5 PAR Profile Method 3: CE-LEDIF 3.6 Quantitative Analysis of PAR Length Profiles Using the CE-LEDIF Method 4 Notes References Chapter 2: Detecting and Quantifying pADPr In Vivo 1 Introduction 2 Materials 2.1 Detecting Poly-ADP-Ribosylated Proteins by Western Blotting 2.2 Detecting pADPr by Immunohistochemistry 2.3 Double Immunofluorescent Labeling of PARP-1 and pADPr 2.4 Quantifying pADPr by Enzyme-Linked Immunoassays (ELISA) 2.5 Identification of Poly-ADP-Ribosylated Proteins Using Small-Scale Two-Dimensional Electrophoresis 3 Methods 3.1 Detecting Poly-ADP-Ribosylated Proteins by Western Blotting 3.1.1 SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE) 3.1.2 pADPr Western Blotting 3.2 Detecting pADPr by Immunohistochemistry 3.2.1 Cryopreserved Brain Preparation 3.2.2 Colorimetric Immunohistochemistry 3.3 Double Immunofluorescent Labeling of PARP-1 and pADPr in Paraffin-Embedded Section 3.3.1 Antigen Retrieval of Paraffin-Embedded Sections 3.3.2 Double Immunofluorescent Labeling of PARP-1 and pADPr 3.3.3 From This Point Forward, Minimize Light Exposure 3.4 Quantifying pADPr by Enzyme-Linked Immunoassay (ELISA) 3.4.1 pADPr Standard 3.4.2 pADPr ELISA (See Note 10) 3.5 Small-Scale Two-Dimensional (2D) Electrophoresis of Poly-ADP-Ribosylated Proteins 3.5.1 Immunoprecipitation of the Poly-ADP-Ribosylated Proteins 3.5.2 Isoelectric Focusing (See Note 14) 3.5.3 Equilibrating IPG Strips 3.5.4 SDS-PAGE Electrophoresis 3.5.5 Protein Visualization Using Sypro Ruby (See Note 17) 4 Notes References Chapter 3: Quantitative Analysis of Nuclear Poly(ADP-Ribose) Dynamics in Response to Laser-Induced DNA Damage 1 Introduction 2 Materials 2.1 Plasmid Purification 2.2 Preparation of the LivePAR, XRCC1-EGFP, EGFP-Polβ, and Control Lentivirus 2.3 Development of Cell Lines Expressing Fluorescently Tagged Genes 2.4 Validation of Transgene Expression by Immunoblot 2.5 Validation of Transgene Expression by Confocal Microscopy 2.6 Laser-Induced DNA Damage 2.7 PAR Immunostaining of DNA Damage-Induced Foci 2.8 Recruitment Dynamic Analysis 3 Methods 3.1 Plasmid Purification 3.2 Preparation of the LivePAR, XRCC1-EGFP, EGFP-Polβ, and Control Lentivirus 3.3 Development of Cell Lines Expressing Fluorescently Tagged Genes 3.4 Validation of Transgene Expression by Immunoblot 3.5 Validation of Transgene Expression by Confocal Microscopy 3.6 Laser-Induced DNA Damage 3.7 PAR Immunostaining of DNA Damage-Induced Foci 3.8 Recruitment Dynamic Analysis 4 Notes References Chapter 4: Analyzing PARP1 Activity: Small Molecule Reactants and Attached Chains of Poly (ADP-Ribose) 1 Introduction 2 Materials 2.1 Labware 2.2 Buffers and Solutions 2.3 Proteins, DNA, and Substrate 2.4 Instruments 3 Methods 3.1 PARP1 Reactions 3.2 Centrifugation and Separation of Small Molecules from Proteins 3.3 Reneutralizations 3.4 PAR Chain Removal 3.5 HPLC Conditions 4 Notes References Chapter 5: Detecting Poly (ADP-Ribose) In Vitro and in Cells Using PAR Trackers 1 Introduction 2 Materials 2.1 Using PAR-T ddGFP in Biochemical Assays 2.1.1 Materials for the Purification of Recombinant PAR-T ddGFP Proteins 2.1.2 Materials for In Vitro Auto(ADP-Ribosyl)ation Reactions with PARP1 2.1.3 Materials for ELISA Assays with Free PAR 2.2 Using PAR-T ddGFP in Cell-Based Assays 2.2.1 Materials for the Expression of PAR-T Sensors in Cells 2.2.2 Materials for Live Cell Imaging Using PAR-T ddGFP 2.2.3 Materials for Luciferase Assays Using PAR-T NanoLuc 3 Methods 3.1 Using PAR-T ddGFP in Biochemical Assays 3.1.1 Purification of Recombinant PAR-T ddGFP Proteins 3.1.2 In Vitro Auto(ADP-Ribosyl)ation Reactions with PARP1 3.1.3 ELISA Assays with Free PAR or PAR-Containing Samples 3.2 Using PAR-T ddGFP in Cellular Assays 3.2.1 Expression of PAR-T Sensors in Cells 3.2.2 Single Cell Imaging Using PAR-T ddGFP 3.2.3 Luciferase Assays Using PAR-T NanoLuc 3.3 Considerations and Limitations When Using the PAR-T Sensors 4 Notes References Chapter 6: An Enzyme-Linked Immunosorbent Assay to Quantify Poly (ADP-Ribose) Level In Vivo 1 Introduction 2 Materials 2.1 Sample Preparation 2.2 ELISA 3 Methods 3.1 Sample Preparation 3.2 Double-Antibody Sandwich ELISA for PAR 4 Notes References Chapter 7: Subcellular Quantitation of ADP-Ribosylation by High-Content Microscopy 1 Introduction 2 Materials 3 Methods 3.1 Seeding Cells 3.2 Treatments 3.3 Fixation, Permeabilization, and Blocking 3.4 Immunostaining 3.5 DAPI and Mounting 3.6 Analysis and Quantification 4 Notes References Chapter 8: A Simple Method to Study ADP-Ribosylation Reversal: From Function to Drug Discovery 1 Introduction 2 Materials 2.1 Chemicals 2.1.1 General 2.1.2 Protein/Peptide Substrate Preparation 2.1.3 Preparation of Thymidine-ADP-Ribosylated Oligonucleotides 2.2 Consumables 2.3 Proteins/Nucleic Acids 2.3.1 Required 2.3.2 Assay Dependent 2.4 Buffers and Other Preparations 2.5 Equipment 3 Methods 3.1 Substrate Preparation 3.1.1 ADP-Ribosylated Peptides 3.1.2 Enzymatically Modified PARP1 3.1.3 Thymidine-ADP-Ribosylated Oligonucleotides 3.2 (ADP-Ribosyl)hydrolase Activity Measurements 3.2.1 In-Solution Substrates 3.2.2 Bead-Bound Substrates 3.2.3 High-Throughput Considerations 4 Notes References Part II: Identification of Protein Targets Chapter 9: Immunoprecipitation Using Mono-ADP-Ribosylation-Specific Antibodies 1 Introduction 2 Materials 2.1 Histone Purification 2.2 Immunoprecipitation 2.3 Western Blotting 3 Methods 3.1 Histone Purification 3.2 RIPA Cell Lysis for Whole Cell Lysate Pull-Downs 3.3 Immunoprecipitation 3.3.1 Histone and Peptide Immunoprecipitation 3.3.2 Whole Cell Lysate Immunoprecipitation 3.3.3 Cross-Linking of Antibody 3.4 Western Blotting 4 Notes References Chapter 10: A Clickable NAD+ Analog-Based Assay of Poly(ADP-Ribosyl)ated Proteins 1 Introduction 2 Materials 2.1 PARylation Reaction 2.2 Methanol-Chloroform Precipitation of PARylated Proteins 2.3 Bio-Orthogonal Conjugation of Labeled Proteins to Disulfide Azide Agarose Beads 2.4 Washing and Cleavage of PARylated Proteins 2.5 Immunoblotting 3 Methods 3.1 PARylation Reaction 3.2 Methanol-Chloroform Precipitation of PARylated Proteins 3.3 Bio-Orthogonal Conjugation of Labeled Proteins to Disulfide Azide Agarose Beads 3.4 Washing and Cleavage of PARylated Proteins 3.5 Immunoblotting 4 Notes References Chapter 11: Functional Analysis of Histone ADP-Ribosylation In Vitro and in Cells 1 Introduction 2 Materials 2.1 Expression and Purification of Recombinant Proteins 2.1.1 Materials for the Purification of Recombinant PARP1 and PARP2 Expressed in Insect Cells 2.1.2 Materials for the Purification of Recombinant Histones Expressed in E. coli 2.1.3 Materials for the Purification of Recombinant HPF1 and NMNAT-1 Expressed in E. coli 2.2 In Vitro Histone ADPRylation Assays 2.2.1 Materials for the Assembly of Mononucleosomes by Salt Gradient Dialysis 2.2.2 Materials for In Vitro Histone ADPRylation Reactions 2.2.3 Materials for Analyzing Histone ADPRylation Reactions by Western Blotting 2.3 Cell-Based Histone ADPRylation Assays 2.3.1 Materials for Generating Cell Lines Ectopically Expressing Wild-Type and ADPRylation Site-Mutant Histones 2.3.2 Materials for Confirming the Ectopic Expression of Flag-Tagged Histones in Cells by Western Blotting 2.3.3 Materials for Analyzing Histone ADPRylation by Nucleosome Immunoprecipitation in Cells Ectopically Expressing Histones 2.4 Assays to Assess the Nuclear Effects of Histone ADPRylation 2.4.1 Materials for Analyzing the Ectopic Expression of Flag-Tagged Histones in Cells by Indirect Immunofluorescence 2.4.2 Materials for the Acid Extraction of Histones from Cultured Cells 2.4.3 Materials for Analyzing by Mass Spectrometry the Effects of Histone ADPRylation on Other Histone Modifications in Cells 3 Methods 3.1 Expression and Purification of Recombinant Proteins 3.1.1 Purification of Recombinant PARP1 and PARP2 Expressed in Insect Cells 3.1.2 Purification of Recombinant Histones Expressed in E. coli 3.1.3 Purification of Recombinant HPF1 and NMNAT-1 Expressed in E. coli 3.2 In Vitro Histone ADPRylation Assays 3.2.1 Assembly of Mononucleosomes by Salt Gradient Dialysis 3.2.2 In Vitro Histone ADPRylation Reactions 3.3 Cell-Based Histone ADPRylation Assays 3.3.1 Preparation of Recombinant Lentiviruses for Ectopic Expression of Histones in Cells 3.3.2 Generation of Mammalian Cell Lines Stably Expressing Ectopic Wild-Type or ADPRylation Site-Mutant Histones 3.3.3 Confirming the Ectopic Expression of Flag-Tagged Histones in Cells by Western Blotting 3.3.4 Analyzing Histone ADPRylation by Nucleosome Immunoprecipitation in Cells Ectopically Expressing Histones 3.4 Assays to Examine the Effects of Histone ADPRylation on Chromatin 3.4.1 Analyzing the Ectopic Expression of Flag-Tagged Histones and Effects on Chromatin Endpoints in Cultured Cells by Indirec... 3.4.2 Analyzing the Effects of ADPRylation on Histone PTMs in Acid-Extracted Histones from Cultured Cells 3.5 Integrating Assays to Study Histone ADPRylation 4 Notes References Part III: Functional Analysis Chapter 12: Studying the Immunomodulatory Functions of PARP1 and PARP2 in Mouse Models of Cancer 1 Introduction 2 Materials 2.1 Syngeneic Mouse Tumor Cell Line Culture and Implantation in Mice 2.1.1 Tissue Culture 2.1.2 Implantation and Follow-Up Tumor Progression 2.2 Necropsy 2.3 Identification of Immune Cells in Tumors by Immunohistochemistry 2.3.1 Tissue Fixation with 10% Neutral-Buffered Formalin 2.3.2 Processing, Paraffin Embedding, and Sectioning 2.3.3 Immunohistochemical Labeling of Immune Cells in Formalin-Fixed and Paraffin-Embedded Tumor Tissues 2.3.4 Visualization of Positive Immunolabeling and Quantitative Evaluation of Immune Subpopulations in Tumor Tissues 2.4 Analysis of Immune Cell Infiltrates by Flow Cytometry 2.4.1 Harvesting of Tumor-Infiltrating Immune Cells 2.4.2 Cell Surface Staining 2.4.3 Intracellular Cell Staining 2.4.4 FACS Acquisition and Analysis 3 Methods 3.1 Syngeneic Mouse Tumor Cell Line Culture and Implantation in Mice 3.1.1 Tissue Culture 3.1.2 Implantation and Follow-Up Tumor Progression 3.2 Necropsy 3.3 Identification of Immune Cells in Tumors by Immunohistochemistry 3.3.1 Tissue Fixation with 10% Neutral-Buffered Formalin 3.3.2 Processing, Paraffin Embedding, and Sectioning Processing Paraffin Embedding Sectioning 3.3.3 Immunohistochemical Labeling of Immune Cells in Formalin-Fixed and Paraffin-Embedded Tumor Tissues 3.3.4 Visualization of Positive Immunolabeling and Quantitative Evaluation of Immune Subpopulations in Tumor Tissues 3.4 Analysis of Immune Cell Infiltrates by Flow Cytometry 3.4.1 Harvesting of Tumor-Infiltrating Immune Cells 3.4.2 Cell Surface Staining 3.4.3 Intracellular Cell Staining 3.4.4 FACS Acquisition and Analysis 4 Notes References Chapter 13: Methods for Investigating Transient Receptor Potential Melastatin-2 (TRPM2): A Cation Channel Activated by ADP-Rib... 1 Introduction 2 Materials 2.1 Quantification of Intracellular Calcium Release 2.2 Subcellular Fractionation Methods for Cellular Localization Analysis 2.3 Knockdown of TRPM2 Protein Expression by RNA Interference 2.4 Preparation of Samples for Detection of TRPM2 by Western Blotting 2.5 Western Blotting for TRPM2 3 Methods 3.1 Measurement of Intracellular Calcium Influx via Activation of TRPM2 3.2 Cellular Localization Analysis of TRPM2 by Subcellular Fractionation 3.2.1 Subcellular Fractionation by Dounce Homogenization 3.2.2 Subcellular Fractionation by Vortexing 3.2.3 Subcellular Fractionation Using a Commercially Available Kit 3.3 Knockdown of TRPM2 Protein Expression by RNA Interference 3.4 Preparation of Samples for Detection of TRPM2 by Western Blotting 3.5 Western Blotting for TRPM2 4 Notes References Chapter 14: Methods to Assess the Role of PARPs in Regulating Mitochondrial Oxidative Function 1 Introduction 2 Materials 2.1 Cell Culture 2.2 PARP Inhibition 2.2.1 Virus and Plasmid-Based Gene Silencing 2.2.2 Pharmacological Inhibition 2.3 Quantitative RT-PCR 2.4 Measurement of the Mitochondrial Membrane Potential and the Superoxide Production Using Flow Cytometry 2.5 Measurement of Adenosine 5′-Triphosphate (ATP) Content of Cells Using ATP Bioluminescent Assay Kit 2.6 Determination of the Mitochondrial DNA/Genomic DNA Ratio 2.7 Oximetry Using the Seahorse XF 96 Instrument 2.8 Assessment of Mitochondrial Morphology 2.9 Isolation and Culture of hADMSCs 2.10 White Adipocyte Differentiation 2.11 Beige Adipocyte Differentiation 3 Methods 3.1 Virus-Based Gene Silencing 3.2 shRNA-Based Silencing 3.3 Quantitative RT-PCR 3.4 Measurement of the Mitochondrial Membrane Potential and the Superoxide Production by Flow Cytometry 3.5 Measurement of Adenosine 5′-Triphosphate (ATP) Content of Cells 3.6 Determination of Mitochondrial DNA 3.7 Determination of Mitochondrial Oxidation Using the Seahorse XF 96 Instrument 3.7.1 Procedures on the Day Before the Assay 3.7.2 Procedures on the Day of the Assay 3.8 Measurement of Mitochondrial Morphology 3.9 Isolation of hADMSCs from Adipose Tissue 3.10 White Adipocyte Differentiation 3.11 Beige Adipocyte Differentiation 4 Notes References Chapter 15: Characterizing ADP-Ribosylation Sites Using Af1521 Enrichment Coupled to ETD-Based Mass Spectrometry 1 Introduction 2 Materials and Equipment 2.1 Cell Culture and Lysis 2.2 In-Solution Protein Digestion 2.3 Purification of Peptides on Sep-Pak 2.4 GST-Protein Expression and Purification of Af1521 2.5 Reduction of ADP-Ribose Polymers 2.6 Enrichment and Purification of ADP-Ribosylated Peptides 2.7 Purification and Desalting of ADP-Ribosylated Peptides for MS Analysis with Stage-Tip Fractionation 2.8 Mass Spectrometric Analysis of ADP-Ribosylated Peptides 3 Methods 3.1 Cell Culture and Lysis 3.2 In-Solution Protein Digestion 3.3 Purification of Peptides on Sep-Pak 3.4 GST-Protein Expression and Purification of Af1521 3.5 Reduction of ADP-Ribose Polymers 3.6 Enrichment and Purification of ADP-Ribosylated Peptides 3.7 Purification and Desalting of ADP-Ribosylated Peptides by StageTip Fractionation 3.8 Mass Spectrometric Analysis of ADP-Ribosylated Peptides 3.9 Data Analysis 3.10 Data Filtering 4 Notes References Chapter 16: Cytological Approaches to Visualize Intracellular Dynamics of RNA-Binding Proteins at Active Genes in Drosophila 1 Introduction 2 Materials 2.1 Equipment and Other Items 2.2 Fly Culture 2.3 Polytene Chromosome Squash Preparation 2.4 Immunostaining 2.5 Fluorescent RNA:RNA In Situ Hybridization (FRISH) 3 Methods 3.1 Polytene Chromosome Squash Preparation and Immunostaining 3.2 Fluorescent RNA In Situ Hybridization Using Intact Tissues (FRISH) 3.2.1 Designing and Synthesis of Riboprobe 3.2.2 Pre-treatment of Tissue 3.2.3 Probe Hybridization 3.2.4 Co-immunostaining and Detection of RNA:RNA Hybrids In Situ 3.3 FRISH on Polytene Chromosome Squash Preparations 3.4 Live Cell Imaging Using Fluorescent Reporter Tagged Proteins 3.4.1 Live Cell Imaging of GFP-Tagged RBPs to Examine Their Binding Patterns at Different Gene Loci in Polytene Nuclei of Dros... 3.4.2 Fluorescence Recovery After Photobleaching (FRAP) 3.4.3 Fluorescence Loss in Photobleaching (FLIP) 4 Notes References Part IV: Poly(ADP-Ribosyl)ating Pathway in Chromatin and Gene Expression Chapter 17: Chromatin Immunoprecipitation Approach to Determine How PARP1 Domains Affect Binding Pattern to Chromatin 1 Introduction 2 Materials 2.1 Drosophila Lines 2.2 Nuclear Isolation 2.3 ChIP Assay 2.4 ChIP PCR Control 2.5 Preparation of the Sequencing Library 2.6 Reads Mapping and ChIP-Seq Analysis 3 Methods 3.1 Preparation of Samples for a Whole Organism ChIP at Third Instar Larvae 3.2 (Optional) Preparation of Samples with Nuclear Isolation 3.3 Samples Cross-Linking and Shearing 3.4 (Optional) Check of DNA Fragment Sizes 3.5 Chromatin Immunoprecipitation 3.6 DNA Isolation 3.7 qPCR to Check the Specific Binding of the Different Version of PARP1 3.8 Preparation of the Sequencing Library 3.8.1 End Repair 3.8.2 Adaptor Ligation 3.8.3 Cleanup of Adaptor-Ligated DNA 3.8.4 PCR Enrichment of Adaptor-Ligated DNA 3.8.5 Cleanup of PCR Reaction 3.9 Sequencing 3.10 Reads Mapping and ChIP Analysis 4 Notes References Chapter 18: Approach to Measuring the Effect of PARP1 on RNA Polymerase II Elongation Rates 1 Introduction 2 Materials 2.1 Buffer Stocks 2.2 Culture of S2 Drosophila Cells 2.3 PARP1 Knockdown 2.4 PARylation Inhibition 2.5 Transcription Inhibition 2.6 Nascent RNA Labeling 2.7 Total RNA Extraction 2.8 Biotinylation of Labeled RNA 2.9 Separation of Nascent RNA 2.10 Gel Electrophoresis 3 Methods 3.1 SiRNA Preparation for PARP1 Knockdown in S2 Drosophila Cell Line 3.2 PARP1 Knockdown in S2 Drosophila Cells 3.3 PARylation Inhibition 3.4 Transcription Inhibition (Synchronize Cell Transcription Before Nascent RNA Labeling) 3.5 4-Thiouridine (4sU) Labeling of Nascent mRNA 3.6 Extraction of Total RNA 3.7 Nascent RNA Biotinylation 3.8 Bead Preparation and 4sU-Tagged RNA Enrichment 3.9 Reverse Transcription to Validate Enrichment of Nascent mRNA Prep by qRT-PCR 3.10 Library Preparation and Parallel Sequencing 4 Notes References Chapter 19: Examining the Effect of PARP-1 Inhibitors on Transcriptional Activity of Androgen Receptor in Prostate Cancer Cells 1 Introduction 2 Materials 3 Methods 3.1 Cell Preparation 3.2 Cell Transduction 3.3 Testing the Effect of PARP-1 Inhibitors on the AR Transcriptional Activity in PC Cells 4 Notes References Part V: Using Animal Models Chapter 20: Using Drosophila Genetics to Identify Factors that Affect PARP1 Activity In Vivo 1 Introduction 2 Materials 2.1 Drosophila Lines 2.2 Salivary Gland Dissection and Culturing 2.3 Western Blot 2.4 Immunostaining 3 Methods 3.1 Parg27.1 Genetic Specificities 3.2 Larva Preparation, Salivary Gland Dissection, and Incubation 3.2.1 Larva Preparation and Selection 3.2.2 Fly Extract Preparation 3.2.3 Salivary Gland Dissection and Incubation 3.3 pADPr Level Measurement by Western Blot 3.3.1 Protein Migration 3.3.2 Wet Membrane Transfer 3.3.3 Blocking 3.3.4 Antibody Incubation and Revelation 3.3.5 (Optional) Stripping 3.4 Immunostaining 4 Notes References Chapter 21: Generating PARP Knockout D. melanogaster with CRISPR/Cas9 System 1 Introduction 2 Materials 2.1 gRNA Design 2.2 gRNA Expressing Plasmid Cloning 2.3 Fly Crosses 2.4 Mutations Detection 3 Methods 3.1 gRNA Design 3.2 gRNA Expressing Plasmid Cloning 3.3 Fly Crosses to Generate PARP Mutant Stocks 3.4 Mutations Detection 4 Notes References Chapter 22: TaqMan Multiplex qPCR Method to Genotype PARG Knockout Mice 1 Introduction 2 Materials 2.1 DNA Isolation from Embryos or Other Tissues 2.2 DNA Isolation from Mice Tissues 2.3 Multiplex Reaction to Detect Knockout Allele Using qPCR 3 Methods 3.1 DNA Isolation from Embryos 3.2 DNA Isolation from Mouse Tissues 3.3 Multiplex Reaction to Detect Knockout Allele Using qPCR 3.4 Multiplex Reaction to Evaluate Cre Recombinase Efficiency in Conditional Knockout 4 Notes References Part VI: Designing and Testing PARP-1 Inhibitors Chapter 23: Cell-Based Screening for New PARP Inhibitors Utilizing PARG-Mutated Mouse Embryonic Stem Cells 1 Introduction 2 Materials 2.1 Cell Seeding and Growing with Tested Compounds 2.2 Fixing and Staining Cells for pADPr 2.3 Cell Imaging and Analysis 2.4 Flow Cytometry Analysis of pADPr Intensity 3 Methods 3.1 Cell Seeding and Growing with Tested Compounds 3.2 Fixing and Staining Attached Cells for pADPr 3.3 Cell Imaging and Analysis 3.4 Flow Cytometry Analysis of pADPr Intensity 4 Notes References Chapter 24: Quantification of PARP7 Protein Levels and PARP7 Inhibitor Target Engagement in Cells Using a Split Nanoluciferase... 1 Introduction 2 Materials 3 Methods 3.1 Nanoluciferase Assay 3.2 Data Processing 4 Notes References Part VII: Targeting the Understudied Components of pADPr Pathway Chapter 25: Purification of Recombinant Human PARG and Activity Assays 1 Introduction 2 Materials 2.1 Materials for Bacterial Growth and Protein Expression 2.2 Materials for Purification of GST-hPARG 2.3 Materials for PreScission Cleavage 2.4 Materials for Large-Scale PAR Synthesis 2.5 Materials for SDS-PAGE 2.6 Materials for Western Blot 2.7 Detection of PARG Activity in Solution 2.8 Materials for Electrophoresis on Sequencing Gel 2.9 Materials for Detection of PARG Activity on Dot Blot 3 Methods 3.1 Purification Protocol Overview 3.2 Bacterial Growth and Induction of Expression 3.3 Cell Lysate Preparation 3.4 Glutathione Sepharose 4B Affinity Chromatography 3.5 PreScission Protease Cleavage 3.6 Quality Control: SDS-PAGE 3.7 Quality Control: Western Blot 3.8 Detection of PARG Activity by Electrophoresis on Sequencing Gel 3.8.1 Purification of Radioactive Poly (ADP-Ribose) 3.8.2 PARG Activity in Solution 3.8.3 Electrophoresis on Sequencing Gel Preparation of Glass Plates 3.8.4 Pouring the Gel 3.8.5 Pre-electrophoresis 3.8.6 Electrophoresis 3.8.7 PARG Activity Dot Blot and Immuno-Detection of PAR 4 Notes References Chapter 26: Purification of Recombinant Human PARP-3 1 Introduction 2 Materials 2.1 Materials for Bacterial Growth and Protein Expression 2.2 Materials for Purification of PARP-3 2.3 Materials for SDS-PAGE 2.4 Materials for Western Blot 2.5 Materials for Detection of PARP Activity with Biotin-NAD+ 2.6 Materials for Detection of PARP Activity with Radioactivity [32P]-NAD+ 3 Methods 3.1 Purification Protocol Overview 3.2 UNICORN Methods Controlling the A KTA Purifier System 3.3 3-AB ECH-Sepharose 4B Column Synthesis 3.3.1 Preparing the Matrix 3.3.2 3-AB Coupling Reaction 3.3.3 3-AB Coupling Procedure 3.4 Packing Sepharose 3.5 Bacterial Growth and Expression Induction 3.6 Cell Lysate Preparation 3.7 3-AB ECH-Sepharose Affinity Chromatography 3.8 MonoQ Chromatography 3.9 Quality Control: SDS-PAGE 3.10 Quality Control: Western Blot 3.11 Detection of PARP-3 Activity by Western Blot Using Anti-Poly(ADP-Ribose) 3.12 Detection of PARP-3 Activity with Biotin-NAD+ 3.13 Detection of PARP-3 Activity with Radioactivity 4 Notes References Index