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We have developed a protocol which facilitates the isolation of nuclei containing mitotic spindles from the plasmodium of Physarum polycephalum. The nuclei are isolated in a spindle stabilization buffer (SSB) consisting of -4 M glycerol, 5 mM EGTA, 10 mM MgCl2, 10 mM PIPES and 0.1% (v/v) Triton X-100 at pH 6.5. Isolation at 20 degrees C, chelation of Ca2+ and the inclusion of the 4 M glycerol were found to be essential for stabilization of the spindle microtubules. The method allows the isolation of clean nuclei in a reasonable yield. Nuclei were isolated at each of the main stages of mitosis (prophase, metaphase and anaphase) and transmission electron microscopy of the isolated nuclei revealed well preserved arrangements of spindle microtubules which were characteristic of the particular stage of mitosis.

Type

Journal article

Journal

Eur J Cell Biol

Publication Date

05/1984

Volume

34

Pages

52 - 59

Keywords

Cell Nucleus, Electrophoresis, Polyacrylamide Gel, Microscopy, Electron, Mitosis, Molecular Weight, Nucleoproteins, Physarum