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Journal of Cell Science, Vol 66, Issue 1 265-275, Copyright © 1984 by Company of Biologists
JOURNAL ARTICLES |
R Kuriyama, G Keryer and GG Borisy
Mitotic spindles from Chinese hamster ovary (CHO) cells were isolated and purified by a one-step procedure in an isolation medium containing the microtubule-stabilizing drug, taxol. Released mitotic spindles were examined by phase-contrast, polarizing and differential-interference microscopy. They were also stained with monoclonal antibody raised against yeast tubulin and examined by epifluorescence microscopy. The spindles were free from visible cytoplasmic contaminants and the chromosomes were generally lost from the preparations. Electron microscopy showed that microtubules were the dominant structural component and sodium dodecyl sulphate/gel electrophoresis showed that tubulin was the major molecular species present, although a number of minor components, possibly representing microtubule-associated proteins (MAPs), were present. The taxol procedure was also useful in obtaining other microtubule-containing structures such as the midbody or the cytoplasmic microtubule complex in interphase cells. The taxol procedure was also used to isolate mitotic spindles from HeLa cells. The HeLa spindles stained positively with an antibody specific for the 210 X 10(3) Mr microtubule-associated protein, indicating that the MAP was retained by the taxol procedure. The taxol procedure appears to be of great advantage in large-scale preparations of spindles for biochemical analysis.
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