Mitosis and Meiosis by Conly L. Rieder (Eds.)

By Conly L. Rieder (Eds.)

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Isolation of Centrosomes from Activated Oocyte Lysates A. Materials and Equipment B. Procedure VII. Immunofluorescence of Centrosomes and Asters A. Preparation of Elvanol Mounting Medium 13. Immunofluorescence of Asters Assembled in Clarified Spisula Oocyte Lysate C. Immunofluorescence of Asters Reconstituted in Vitro VII. Electron Microscopy of Asters and Centrosomes A. Comments IX. Summary References 35 Robert E. Palazzo and Jacalyn M. Vogel 36 I. Introduction Most animal cells contain a single centrosome that functions as the major microtubule organizing center.

22-pm filter, except for sucrose solutions. which must be sterilized with an autoclave at 105°C. 2. Procedure 1. The human lymphoblastic KE37 cells are cultured in suspension in RPMI 1640 medium supplemented with 7% of fetal calf serum (FCS) at 37°C and 5% C02. Exponentially growing cells (8 X los to 1 X lo6 cells/ml) are incubated for 1 hr with 2 X lo-’ M nocodazole at 37°C. 2. Cells, classically from 1 to 3 liters, are washed by rapid centrifugation and resuspended at 4°C once with half of the initial volume of TBS buffer and a second time with half volume of TBS 1/10 8% sucrose buffer.

This is also the case for other major proteins, including G3PDH (1). Tcpl (2), hsp70 (3), hsp90 (4), and HsEg5 (5). the control of the nucleation of microtubules, and undergoes a duplication process once per cell division cycle. That such properties are apparently maintained among divergent species suggests that functional protein complexes are also conserved. This is the case for y-tubulin (Oakley, 1992), which has been discovered in a genetic screen for revertants of P-tubulin mutation in Aspergillus nidulans and has been involved in microtubule nucleation (for review, see Erickson and Stoeffer, 1996; Pereira and Schiebel, 1997).

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