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Related Experiment Videos

Isolation of centrosomes from Spisula solidissima oocytes

R E Palazzo1, J M Vogel

  • 1Department of Biochemistry, Cell, and Molecular Biology, University of Kansas, Lawrence 66045, USA.

Methods in Cell Biology
|January 19, 1999
PubMed
Summary

This study presents a method for isolating large quantities of centrosomes from surf clam oocytes, enabling year-round biochemical and structural analysis of microtubule nucleation and centrosome composition.

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Area of Science:

  • Cell Biology
  • Molecular Biology
  • Biochemistry

Background:

  • Centrosomes are crucial for microtubule organization and cell division.
  • Previous methods for centrosome isolation were limited in scale and accessibility.
  • Understanding centrosome composition and function requires robust biochemical and structural analysis techniques.

Purpose of the Study:

  • To describe a scalable method for isolating centrosomes from surf clam (S. solidissima) oocytes.
  • To enable year-round biochemical and structural studies of centrosomes.
  • To facilitate the identification of novel centrosome proteins and elucidate molecular mechanisms of centrosome function.

Main Methods:

  • Parthenogenetic activation of surf clam oocytes.
  • Preparation of oocyte lysates and storage at -80°C.

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  • Sucrose-density gradient centrifugation for centrosome purification (3000–4000-fold).
  • Storage of isolated centrosomes in high sucrose media at -80°C.

    • Main Results:

    • Scalable preparation of up to 2 liters of lysate per person during oocyte availability (June-August).
    • Obtained 2–3 x 10^6 centrosomes per ml of frozen lysate with high purity.
    • Isolated centrosomes retain microtubule nucleation potential after long-term storage.
    • Yielded 420–600 micrograms of centrosome protein daily, with potential for 20–40 mg annually per individual.
    • Provided sufficient centrosome protein for antibody generation and protein sequencing.

    Conclusions:

    • The surf clam oocyte system provides a convenient and scalable source for centrosome isolation.
    • This method supports combined structural and biochemical approaches for centrosome research.
    • Enables detailed analysis of centrosome composition, microtubule nucleation, and assembly mechanisms.