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Protein Modification to Probe Intradynein Interactions and In Vivo Redox State

  • Ken-ichi Wakabayashi
  • Miho Sakato
  • Stephen M. King
Part of the Methods in Molecular Biology™ book series (MIMB, volume 392)

Abstract

Dyneins are highly complex molecular motors containing multiple components that contribute motor, regulatory and cargo-binding activities. Within cilia/flagella, these enzymes comprise the inner and outer arms associated with the doublet microtubules. In this chapter, we describe how to purify the outer dynein arm from flagella of the unicellular green alga Chlamydomonas, which is one of the best characterized members of this motor class. We also detail the methods that we use to identify interactions involving dynein components by chemical cross-linking and a recently developed technique to assess the in vivo redox state of thioredoxin-like proteins that are associated with axonemal dyneins from a wide range of organisms. Finally, we describe how to purify highly specific antibodies from serum by blot purification using recombinant proteins. Although designed for analysis of Chlamydomonas flagellar dyneins, these approaches should be readily adaptable to the study of other systems.

Key Words

Chlamydomonas cilia cross-linking dynein flagella redox poise thioredoxin 

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Copyright information

© Humana Press Inc., Totowa, NJ 2007

Authors and Affiliations

  • Ken-ichi Wakabayashi
    • 1
    • 2
  • Miho Sakato
    • 2
  • Stephen M. King
    • 2
  1. 1.Department of Biological Sciences, Graduate School of ScienceUniversity of TokyoTokyoJapan
  2. 2.Department of Molecular, Microbial, and Structural BiologyUniversity of Connecticut Health CenterFarmington

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