00000nmm a2200000 i 4500 UP-100048679771077598 Buklod 20230525163342.0 m o u cr ||||||||||| 230525s2013 cau go u eng d 9780123979452 (OCoLC)840880365 OCLCO DSCI rda eng DMLUC QP 310 C5 C55 2013eb Cilia part A edited by Wallace F. Marshall. San Diego, CA Elsevier Science [2013] 1 online resource (397 pages) illustrated (some color) text rdacontent computer rdamedia online resource rdacarrier Methods in enzymology volume 524 Includes bibliographical references and index. Front Cover; Cilia, Part A; Copyright; Contents; Contributors; Preface; Methods in Enzymology; Chapter One: Isolation of Intraflagellar Transport Particle Proteins from Chlamydomonas reinhardtii; 1. Introduction; 2. Media; 2.1. Tips on making media; 2.2. Media choices; 3. Isolating Flagella; 3.1. Recipe of stock solutions (the chemicals are from Sigma); 3.2. Preparing the culture; 3.3. Isolating flagella; 4. Preparing Soluble Flagellar Proteins; 5. Isolating IFT Particle Proteins by Fractioning Soluble Flagellar Proteins Through Sucrose Density Gradient. Isolation of Intraflagellar Transport Particle Proteins from Chlamydomonas reinhardtii -- Molecular Tools for Studying the Radial Spoke -- Methods for Analysis of Calcium/Calmodulin Signaling in Cilia and Flagella -- Analysis of Flagellar Protein Ubiquitination -- Analysis of Ciliary Import -- Analysis of Signaling Pathways Controlling Flagellar Movements in Mammalian Spermatozoa -- Analysis of Microtubule Plus-End-Tracking Proteins in Cilia -- Biochemical and Physiological Analysis of Axonemal Dyneins -- Measuring the Regulation of Dynein Activity During Flagellar Motility -- Analysis of Interactions Between Intraflagellar Transport Proteins -- Analysis of Ciliary Membrane Protein Dynamics Using SNAP Technology -- Engineering Oscillating Microtubule Bundles -- Purification of Exosome-Like Vesicles from Urine -- Electron Microscopy of Flagella, Primary Cilia, and Intraflagellar Transport in Flat-Embedded Cells -- In vivo Imaging of IFT in Chlamydomonas Flagella -- Measuring Rates of Intraflagellar Transport Along Caenorhabditis elegans Sensory Cilia Using Fluorescence Microscopy -- 3D Structural Analysis of Flagella/Cilia by Cryo-Electron Tomography -- Electron Tomography of IFT Particles -- Reconstitution of Flagellar Sliding. 3.2. Identifying calmodulin-binding proteins3.2.1. Affinity purification using calmodulin-coupled sepharose; 3.2.2. Calmodulin gel overlay assay; 3.2.2.1. Materials; 3.2.2.2. Method; 3.2.3. Calmodulin immunoprecipitation; 3.2.3.1. Materials; 3.2.3.2. Method; 4. Calcium-Induced Modulation of Motility; 4.1. Inducing and imaging phototaxis and photoshock in C. reinhardtii; 4.2. Analyzing photo-accumulation; 4.3. Reactivation studies using cell models or isolated axonemes; 4.4. Microtubule sliding assay; 4.4.1. Materials; 4.4.2. Method; 5. Summary; References. 5.1. Add a tag DNA to a gene5.2. Fuse a short DNA fragment to a tag DNA; 6. Ni-NTA Affinity Purification of the Radial Spoke Complex; 7. Flagella Miniprep; Solutions; 8. Summary; Acknowledgments; References; Chapter Three: Methods for Analysis of Calcium/Calmodulin Signaling in Cilia and Flagella; 1. Introduction; 2. Calcium Entry into the Intraflagellar Space; 3. Calcium-Binding Proteins; 3.1. Identifying calcium sensors by molecular and biochemical methods; 3.1.1. Electrophoretic mobility shift and phenyl-sepharose binding; 3.1.2. Radioisotope 45-Ca2+ 5.1. Prepare low- and high-density sucrose solutions5.2. Making the gradient; 5.3. Gradient centrifugation; 5.4. Collecting the gradient fractions; 5.5. Analyzing the fractions by gel electrophoresis; 6. Isolating IFT Particle Proteins by Immunoprecipitation from Soluble Flagellar Proteins; 7. Conclusions; Acknowledgments; References; Chapter Two: Molecular Tools for Studying the Radial Spoke; 1. Introduction; 2. Recovery of Genomic DNA; 2.1. PCR genomic DNA; 2.2. Random subcloning of BAC DNA; 3. Slot Lysis Assay for Large-Scale Screenings; Solutions; 4. DNA Alterations; 5. Tagging. Chapter Four: Analysis of Flagellar Protein Ubiquitination1. Introduction; 2. Summary of Putative Ubiquitin, Ubiquitin-Like Proteins, E1, E2, and E3 in Flagella/Cilia; 3. Determination of the Flagellar Localization of Candidate Proteins with Fluorescence Proteins; 4. Analysis of the Free Ubiquitin in Flagella; 5. In Vitro Ubiquitination System of Flagella; Reagents; Procedures; 6. Analysis of Flagellar Ubiquitination During Flagellar Shortening; Reagents; Procedures; Procedures; 7. Analysis of Flagellar Ubiquitination During Mating; Procedures; 8. Summary; Acknowledgment; References. IP-based subscription, on campus only. Access via Electronic Resources of the UPD University Library Website. Cilia and ciliary motion. Flagella (Microbiology). Cells Motility. Electronic books. Marshall, Wallace F. editor. Methods in enzymology volume 524. Also available remotely for University of the Philippines System via Science Direct through. Click here to access thru Open Athens https://www.sciencedirect.com/bookseries/methods-in-enzymology/vol/524/suppl/C (viewed 05 December 2022) https://www.sciencedirect.com/bookseries/methods-in-enzymology/vol/524/suppl/C FO UPD DSCI QP 310 C5 C55 2013EB Electronic Resource