Appendix 14: Cytoskeleton Isolation

(Jeffery, 1985)

Chaetopterus eggs have a cortical cytoskeleton which contains nearly all the mRNA of the egg, can be easily isolated, and thus should be useful for the analysis of cytoskeleton-mRNA interactions.

N.B. Triton X-100 cannot be used as the detergent for cytoskeleton isolation from these eggs, because cytoskeletons are not preserved in this detergent.

  1. Remove vitelline envelope [Appendix 9].
  2. Wash in extraction buffer (10 mM PIPES, pH 6.8; 300 mM sucrose; 100 mM KCl; 5 mM Mg acetate; 1 mM EGTA containing protease inhibitors).
  3. Extract on ice for 30 min with extraction buffer + 0.5% Nonidet P-40.
  4. Collect by centrifugation.
  5. Wash three or four times in extraction buffer.