Generation of cell-free extracts of Xenopus eggs and demembranated sperm chromatin for the assembly and isolation of in vitro-formed nuclei for Western blotting and scanning electron microscopy (SEM)

This protocol details methods for the generation of cell-free extracts and DNA templates from the eggs and sperm chromatin, respectively, of the clawed toad Xenopus laevis. We have used this system with scanning electron microscopy ( SEM), as detailed herein, to analyze the biochemical requirements and structural pathways for the biogenesis of eukaryotic nuclear envelopes ( NEs) and nuclear pore complexes ( NPCs). This protocol requires access to female frogs, which are induced to lay eggs, and a male frog, which is killed for preparation of the sperm chromatin. Egg extracts should be prepared in 1 d and can be stored for many months at -80 degrees C. Demembranated sperm chromatin should take only approximately 2 - 3 h to prepare and can be stored at -80 degrees C almost indefinitely. The time required for assembly of structurally and functionally competent nuclei in vitro depends largely on the quality of the cell-free extracts and, therefore, must be determined for each extract preparation.