Investigating mitotic spindle assembly and function in vitro using Xenopus laevis egg extracts

Nat Protoc. 2006;1(5):2305-14. doi: 10.1038/nprot.2006.396.


Extracts from Xenopus laevis eggs provide a powerful system for the study of cell division processes in vitro through biochemical reconstitution and manipulation, and microscopic analysis. We provide protocols for the preparation of metaphase-arrested extracts and in vitro assays to examine the following pathways of spindle assembly: 1) Sperm nuclei added to meiotic extracts, supporting the formation of half-spindles and bipolar spindle structures around unreplicated chromosomes; 2) sperm nuclei added to extracts that cycle through interphase and form spindles that are capable of undergoing anaphase and chromosome segregation; and 3) spindle formation around chromatin-coated beads. Finally, we describe methods to inhibit a specific protein by immunodepletion or addition of an inhibitor such as a dominant-negative construct. These techniques can be used to analyze the mitotic function of a given protein. It takes approximately 1.5 h to prepare the extract, 1-3 h for spindle-assembly experiments and an additional 1-3 h if immunodepletion is performed.

MeSH terms

  • Animals
  • Cell Extracts*
  • Cell Nucleus
  • Cell Nucleus Division / physiology*
  • Cytological Techniques*
  • Female
  • Male
  • Ovum*
  • Spermatozoa
  • Spindle Apparatus / physiology*
  • Xenopus laevis


  • Cell Extracts