Description

This new volume of Methods in Enzymology continues the legacy of this premier serial with quality chapters authored by leaders in the field. This volume covers cilia and includes chapters on such topics as electron microscopy of IFT in cilia and flagella, radial spoke isolation and assays, and biomechanical measurements of kinocilium.

Key Features

    • Continues the legacy of this premier serial with quality chapters authored by leaders in the field
    • Covers cilia
    • Contains chapters on such topics as electron microscopy of IFT in cilia and flagella, radial spoke isolation and assays, and biomechanical measurements of kinocilium

    Readership

    Biochemists, biophysicists, molecular biologists, analytical chemists, and physiologists

    Table of Contents

    Series Page

    Contributors

    Preface

    Methods in Enzymology

    Chapter One. Isolation of Intraflagellar Transport Particle Proteins from Chlamydomonas reinhardtii

    Abstract

    1 Introduction

    2 Media

    3 Isolating Flagella

    4 Preparing Soluble Flagellar Proteins

    5 Isolating IFT Particle Proteins by Fractioning Soluble Flagellar Proteins Through Sucrose Density Gradient

    6 Isolating IFT Particle Proteins by Immunoprecipitation from Soluble Flagellar Proteins

    7 Conclusions

    Acknowledgments

    References

    Chapter Two. Molecular Tools for Studying the Radial Spoke

    Abstract

    1 Introduction

    2 Recovery of Genomic DNA

    3 Slot Lysis Assay for Large-Scale Screenings

    4 DNA Alterations

    5 Tagging

    6 Ni-NTA Affinity Purification of the Radial Spoke Complex

    7 Flagella Miniprep

    8 Summary

    Acknowledgments

    References

    Chapter Three. Methods for Analysis of Calcium/Calmodulin Signaling in Cilia and Flagella

    Abstract

    1 Introduction

    2 Calcium Entry into the Intraflagellar Space

    3 Calcium-Binding Proteins

    4 Calcium-Induced Modulation of Motility

    5 Summary

    References

    Chapter Four. Analysis of Flagellar Protein Ubiquitination

    Abstract

    1 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

    6 Analysis of Flagellar Ubiquitination During Flagellar Shortening

    7 Analysis of Flagellar Ubiquitination During Mating

    8 Summary

    Acknowledgment

    References

    Chapter Five. Analysis of Ciliary Import

    Abstract

    1 Intr

    Details

    No. of pages:
    472
    Language:
    English
    Copyright:
    © 2013
    Published:
    Imprint:
    Academic Press
    Print ISBN:
    9780123979452
    Electronic ISBN:
    9780123984869