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).

2.50
Hdl Handle:
http://hdl.handle.net/10541/71362
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).
Authors:
Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; Sanderson, Helen S; Gardiner, Fiona; Kiseleva, Elena; Goldberg, Martin W; Drummond, Sheona P
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.
Affiliation:
Paterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK. tallen@picr.man.ac.uk
Citation:
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). 2007, 2 (5):1173-9 Nat Protoc
Journal:
Nature Protocols
Issue Date:
2007
URI:
http://hdl.handle.net/10541/71362
DOI:
10.1038/nprot.2007.138
PubMed ID:
17546012
Type:
Article
Language:
en
ISSN:
1750-2799
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorAllen, Terence D-
dc.contributor.authorRutherford, Sandra A-
dc.contributor.authorMurray, Stephen M-
dc.contributor.authorSanderson, Helen S-
dc.contributor.authorGardiner, Fiona-
dc.contributor.authorKiseleva, Elena-
dc.contributor.authorGoldberg, Martin W-
dc.contributor.authorDrummond, Sheona P-
dc.date.accessioned2009-06-23T15:49:56Z-
dc.date.available2009-06-23T15:49:56Z-
dc.date.issued2007-
dc.identifier.citationGeneration 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). 2007, 2 (5):1173-9 Nat Protocen
dc.identifier.issn1750-2799-
dc.identifier.pmid17546012-
dc.identifier.doi10.1038/nprot.2007.138-
dc.identifier.urihttp://hdl.handle.net/10541/71362-
dc.description.abstractThis 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.en
dc.language.isoenen
dc.subject.meshAnimals-
dc.subject.meshBlotting, Western-
dc.subject.meshCell Extracts-
dc.subject.meshCell Nucleus-
dc.subject.meshCell-Free System-
dc.subject.meshChromatin-
dc.subject.meshFemale-
dc.subject.meshMale-
dc.subject.meshMicroscopy, Electron, Scanning-
dc.subject.meshOvum-
dc.subject.meshSpermatozoa-
dc.subject.meshXenopus laevis-
dc.titleGeneration 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).en
dc.typeArticleen
dc.contributor.departmentPaterson Institute for Cancer Research, University of Manchester, Wilmslow Road, Withington, Manchester M20 4BX, UK. tallen@picr.man.ac.uken
dc.identifier.journalNature Protocolsen
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