Chapter 3 Isolation of Centrosomes from Spisula solidissima Oocytes

Academic Article

Abstract

  • This chapter describes the methods used for isolating centrosomes from oocytes of the marine surf clam, Spisula solidissima. The oocytes in Spisula are arrested in late prophase of meiosis I and are activated en mass by simply elevating the potassium concentration of the surrounding seawater. Once activated, the oocytes proceed synchronously through a number of critical cell cycle events, including centrosome assembly, germinal vesicle (nuclear envelope) breakdown, and assembly of the meiosis I spindle. The chapter then describes the methods for preparing lysates and obtaining functional centrosomes from oocytes 4 min after their activation. Once isolated, centrosomes can be used for protein analysis, ultrastructural studies, or in functional reconstitution assays. In addition, these preparations offer the isolation of sufficient quantities of centrosome proteins to be used as antigens for generating centrosome-specific antibodies or for obtaining protein sequence for the purpose of antibody production or the design of oligonucleotide primers for isolating cDNA fragments coding for centrosome proteins. © 1999 Academic Press, Inc. All rights reserved.
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    Author List

  • Palazzo RE; Vogel JM
  • Start Page

  • 35
  • End Page

  • 56
  • Volume

  • 61
  • Issue

  • C