Cilia part A - Marshall, Wallace F. (San Diego, CA Elsevier Science, 2013)
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.
Includes bibliographical references and index.
Keywords: Cilia and ciliary motion; Flagella (Microbiology).; Cells -- Motility; Electronic books