Treatment of mouse embryo fibroblasts with 1% Triton X-100 at 37 °C in the presence of 4M glycerol and 1 mM EGTA results in the extraction of about 80% cellular proteins. Indirect immunofluorescent staining with monospecific antibodies against tubulin showed that extracted cultures contained a well developed system of cytoplasmic microtubules, indistinguishable from a system of control non-extracted cells. Microtubules in extracted cells were sensitive to Ca2+ ions, and to cold or prolonged incubation in a glycerol-free buffer. Sodium dodecylsulphate-polyacrilamide gel electrophoresis revealed proteins co-electrophoresed with tubulin and actin in Triton-treated cultures. Electron microscopy demonstrated the presence of both microtubules and microfilament bundles in the extracted cells, but complete dissolution of plasma and intracellular membranes.
ASJC Scopus subject areas
- Cell Biology