title: 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).
abstract: 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.
authors.affiliation: Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK. tallen@picr.man.ac.uk
authors.forename: T D
authors.initials: TD
authors.lastname: Allen
authors.forename: S A
authors.initials: SA
authors.lastname: Rutherford
authors.forename: S
authors.initials: S
authors.lastname: Murray
authors.forename: H S
authors.initials: HS
authors.lastname: Sanderson
authors.forename: F
authors.initials: F
authors.lastname: Gardiner
authors.forename: E
authors.initials: E
authors.lastname: Kiseleva
authors.forename: M W
authors.initials: MW
authors.lastname: Goldberg
authors.forename: S P
authors.initials: SP
authors.lastname: Drummond
mesh_terms: D000818:Animals; D015153:Blotting, Western; D002457:Cell Extracts / Q000302:isolation & purification*; D002467:Cell Nucleus / Q000648:ultrastructure*; D002474:Cell-Free System / Q000648:ultrastructure*; D002843:Chromatin / Q000302:isolation & purification / Q000648:ultrastructure*; D005260:Female; D008297:Male; D008855:Microscopy, Electron, Scanning; D010063:Ovum / Q000166:cytology*; D013094:Spermatozoa / Q000166:cytology*; D014982:Xenopus laevis
chemical_list: D002457:Cell Extracts; D002843:Chromatin