Sperm flagella and axonemes from C. intestinalis were prepared as described previously (33). Sperm were collected from adult sperm ducts. Flagella were isolated via homogenization of sperm in artificial Ca2+-free seawater [462 mM NaCl, 9.39 mM KCl, 59.08 mM MgCl2, 5 mM EGTA, and 10 mM Hepes-NaOH (pH 8.0)], followed by pooling of the supernatant after centrifugation at 1500g for 5 min. The isolated flagella were pelleted by centrifugation at 9100g for 5 min. The axonemes were prepared by demembranation of flagella with 0.05% Triton X-100 in an axoneme buffer [AXB; 150 mM KCl, 1 mM MgSO4, 0.5 mM EGTA, 0.2 mM dithiothreitol, and 20 mM tris-HCl (pH 8.0)], followed by centrifugation.

Isolation of the f/I1 dynein from axonemes was performed following the methods of previous papers (13, 33, 34) with slight modifications. The axonemes were treated with 0.6 M KCl in AXB for 30 min on ice and centrifuged at 17,360g for 15 min. This treatment was repeated twice to remove as much of the OAD as possible. The axonemes were further treated with 0.7 M KCl and 5 mM ATP in AXB for 30 min on ice to extract the f/I1 dynein and centrifuged at 17,360g for 15 min to recover the extract. The extraction of f/I1 dynein was repeated twice. The extracts were subjected to ultracentrifugation at 452,000g for 15 min to remove aggregates before loading onto a sucrose density gradient.

A linear sucrose gradient (5 to 20%) with J-buffer [5 mM MgSO4, 2 mM EGTA, 0.5 mM dithiothreitol, and 20 mM Hepes (pH 7.8)] containing 200 mM potassium acetate was prepared using Gradient Station (BioComp Instruments, Fredericton, NB, Canada). The extracts were loaded onto the linear sucrose gradient and centrifuged in a Himac CP65β centrifuge with a P28S2 rotor (Hitachi, Japan) at 121,930g for 16 hours at 4°C. Fractions of 650 μl were collected. The protein concentrations and the adenosine triphosphatase (ATPase) activity of each fraction were measured using the Bradford method (35) and Taussky and Schorr method (36), respectively, to determine the position of dynein. The fractions containing f/I1 dynein were determined by Western blot analysis using anti–Ci-IC116, anti–Ci-IC110, and anti–Ci-IC105 antibodies.

The sucrose density gradient fractions containing f/I1 dynein were separated on a Uno Q anion exchange column (Bio-Rad, Hercules, CA, USA) using an ÄKTA purifier HPLC system (Amersham Pharmacia Biotech, USA). The column was equilibrated with J-buffer, and the proteins were eluted with a linear gradient of 150 to 450 mM KCl in J-buffer. The fractions containing f/I1 dynein were determined on the basis of ATPase activity and Western blotting results using anti–Ci-IC116, anti–Ci-IC110, and anti–Ci-IC105 antibodies.

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