Isolation of Chlamydomonas Flagella

Branch Craige1, Jason M. Brown1, George B. Witman1

1 Department of Cell and Developmental Biology, University of Massachusetts Medical School, Worcester, Massachusetts
Publication Name:  Current Protocols in Cell Biology
Unit Number:  Unit 3.41
DOI:  10.1002/0471143030.cb0341s59
Online Posting Date:  June, 2013
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Abstract

A simple, scalable, and fast procedure for the isolation of Chlamydomonas flagella is described. Chlamydomonas can be synchronously deflagellated by treatment with chemicals, pH shock, or mechanical shear. The Basic Protocol describes the procedure for flagellar isolation using dibucaine to induce flagellar abscission; we also describe the pH shock method as an Alternate Protocol when flagellar regeneration is desirable. Sub‐fractionation of the isolated flagella into axonemes and the membrane + matrix fraction is described in a Support Protocol. Curr. Protoc. Cell Biol. 59:3.41.1‐3.41.9. © 2013 by John Wiley & Sons, Inc.

Keywords: flagella; flagellar isolation; flagellar regeneration; cilia; axoneme

     
 
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Table of Contents

  • Introduction
  • Support Protocol 1: Fractionation of Isolated Flagella into Axonemes and Membrane + Matrix Fractions
  • Alternate Protocol 1: Flagellar Isolation by the pH Shock Method
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1:

  Materials
  • Cells
  • 10 mM HEPES, pH 7.4 (at room temperature)
  • HMDS‐EGTA (see recipe)
  • HMDS (see recipe)
  • 25 mM dibucaine (see recipe)
  • HMDS‐25% sucrose (see recipe)
  • Sorvall RC‐3B centrifuge with an H‐6000A rotor
  • 250‐ml screw‐cap bottles
  • 50‐ml disposable conical tubes, sterile
  • 10‐ml plastic serological pipets
  • Light microscope
  • 25‐ml serological pipets
  • 50‐ml round‐bottom polycarbonate tubes (Nalgene, cat. no. 3117‐9550)

Support Protocol 1: Fractionation of Isolated Flagella into Axonemes and Membrane + Matrix Fractions

  • Isolated flagella (see Basic Protocol)
  • HMDEKP buffer (see recipe)
  • 10% (v/v) NP‐40 alternative (Calbiochem, cat. no. 492018)

Alternate Protocol 1: Flagellar Isolation by the pH Shock Method

  • 0.5 N acetic acid
  • 0.5 M KOH
  • Small beaker
  • Stir bar and magnetic stirrer
  • pH meter
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Figures

Videos

Literature Cited

Literature Cited
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