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
eBook ISBN:
9780123984869
Print ISBN:
9780123979452

About the serial-volume-editor

Wallace F. Marshall

Wallace Marshall is an electrical engineer by training, who became interested in biology out of a desire to understand how cells solve engineering problems, such as determining the size of organelles. He received his Ph.D. at UCSF with John Sedat, where he studied the diffusional of motion of interphase chromatin using live cell imaging and computational image analysis. He then trained as a postdoc with Joel Rosenbaum at Yale, where he began studying the mechanisms regulating the length of cilia and flagella. He is now Profess of Biochemistry at UCSF, where he lab continues to study the assembly and length regulation of cilia and flagella, as well as the mechanisms that regulate the size of other organelles. His work takes advantage of an integrated combination of methods including genetics, microscopy, and computational modeling, as well as a wide variety of model organisms including Chlamydomonas reinhardtii, Stentor coeruleus, yeast, flatworms, and mammalian cells.

Affiliations and Expertise

Department of Biochemistry & Biophysics, University of California San Fransisco at Mission Bay, USA