Protein modification to probe intradynein interactions and in vivo redox state.
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.
Wakabayashi
Ken-ichi
K
Department of Biological Sciences, Graduate School of Science, University of Tokyo, Tokyo, Japan.
Sakato
Miho
M
King
Stephen M
SM
D000818:Animals; D001369:Axons / Q000378:metabolism; D001671:Biochemistry / Q000379:methods*; D002499:Centrifugation, Density Gradient; D002696:Chlamydomonas / Q000378:metabolism; D002923:Cilia / Q000378:metabolism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D004398:Dyneins / Q000737:chemistry*; D005407:Flagella / Q000378:metabolism; D005786:Gene Expression Regulation; D010084:Oxidation-Reduction; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D040901:Proteomics / Q000379:methods; D013879:Thioredoxins / Q000737:chemistry
D003432:Cross-Linking Reagents; D013879:Thioredoxins; D004398:Dyneins