Full Description
Ataxia-telangiectasia (A-T) is a rare and severe genetic disorder affecting children. A-T is a multisystem disease characterized by progressive neurodegeneration, immunodeficiency and cancer predisposition. This detailed volume explores the ever expanding field of research into the ATM (ataxia-telangiectasia, mutated) gene and the role played by ATM kinase in DNA damage signaling and diverse cellular processes. What follows is a handy desktop reference for both seasoned A-T researchers and postgraduate students, as it demonstrates the breadth of recent developments in A-T studies. 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, readily reproducible laboratory protocols, and tips on troubleshooting and avoiding known pitfalls. Both classic and cutting-edge techniques are described, including ATM gene mutation detection, assays for radiosensitivity and radioresistant DNA synthesis, new methods to measure ATM kinase activity by imaging microscopy and high content screening as well as proteomics, phosphoproteomics and bioinformatics approaches to decipher ATM-dependent signalling pathways. Additional methods include generation of patient-specific stem cells and approaches to study ATM functions in the nervous system.
Comprehensive and practical, ATM Kinase: Methods and Protocols aims to ignite and attract the interest of colleagues from diverse fields to A-T research in an effort to bring their expertise and fresh ideas to resolve many A-T puzzles still waiting to be pieced together and to alleviate the suffering of A-T children and their families.
Contents
Assaying Radiosensitivity of Ataxia-Telangiectasia.- Assaying for Radioresistant DNA Synthesis, the Hallmark Feature of the Intra-S-Phase Checkpoint Using a DNA Fiber Fluorography-Based Methodology.- ATM Gene Mutation Detection Techniques and Functional Analysis.- An HTRF® Assay for the Protein Kinase ATM.- ATM Kinase Inhibitors: HTS Cellular Imaging Assay Using CellomicsTM ArrayScan VTI Platform.- Image-Based High Content Screening: Automating the Quantification Process for DNA Damage-Induced Foci.- Analyzing ATM Function by Electroporation of Endonucleases and Immunofluorescence Microscopy.- Quantitative and Dynamic Imaging of ATM Kinase Activity by Bioluminescence Imaging.- Zn(II)-Phos-Tag SDS-PAGE for Separation and Detection of a DNA Damage-Related Signaling Large Phosphoprotein.- Identification of ATM Protein Kinase Phosphorylation Sites by Mass Spectrometry.- Studies of ATM Kinase Activity Using Engineered ATM Sensitive to ATP Analogues (ATM-AS).- Functional Characterization of ATM Kinase Using Acetylation-Specific Antibodies.- Identification of ATM-Interacting Proteins by Co-Immunoprecipitation and Glutathione-S-Transferase (GST) Pull-Down Assays.- ATM Activation and H2AX Phosphorylation Induced by Genotoxic Agents Assessed by Flow- and Laser Scanning-Cytometry.- Peptide Immunoaffinity Enrichment with Targeted Mass Spectrometry: Application to Quantification of ATM Kinase Phospho-Signaling.- Mass Spectrometry-Based Proteomics for Quantifying DNA Damage-Induced Phosphorylation.- Statistical Analysis of ATM-Dependent Signaling in Quantitative Mass Spectrometry Phosphoproteomics.- ChIP Technique to Study Protein Dynamics at Defined DNA Double-Strand Breaks.- Studies of the DNA Damage Response by Using the Lac Operator/Repressor (LacO/LacR) Tethering System.- Imaging of Fluorescently-Tagged ATM Kinase at the Sites of DNA Double-Strand Breaks.- Live Cell Imaging to Study Real-Time ATM-Mediated Recruitment of DNA Repair Complexes to Sites of Ionizing Radiation-Induced DNA Damage.- Analyzing Heterochromatic DNA Double Strand Break (DSB) Repair in Response to Ionizing Radiation.- Phenotypic Analysis of ATM Protein Kinase in DNA Double-Strand Break Formation and Repair.- Monitoring DNA Repair Consequences of ATM Signaling Using Simultaneous Fluorescent Readouts.- Non-Canonical ATM Activation and Signaling in Response to Transcription-Blocking DNA Damage.- Study of ATM Phosphorylation by Cdk5 in Neuronal Cells.- DNA Damage Response in Human Stem Cells and Neural Descendants.- A Patient-Specific Stem Cell Model to Investigate the Neurological Phenotype Observed in Ataxia-Telangiectasia.- Lentiviral Reprogramming of A-T Patient Fibroblasts to Induced Pluripotent Stem Cells.- Monitoring the ATM-Mediated DNA Damage Response in the Cerebellum Using Organotypic Cultures.
