A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).

2.50
Hdl Handle:
http://hdl.handle.net/10541/70458
Title:
A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).
Authors:
Kiseleva, Elena; Allen, Terence D; Rutherford, Sandra A; Murray, Stephen M; Morozova, Ksenia N; Gardiner, Fiona; Goldberg, Martin W; Drummond, Sheona P
Abstract:
This protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.
Affiliation:
Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia. elka@bionet.nsu.ru
Citation:
A protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM). 2007, 2 (8):1943-53 Nat Protoc
Journal:
Nature Protocols
Issue Date:
2007
URI:
http://hdl.handle.net/10541/70458
DOI:
10.1038/nprot.2007.251
PubMed ID:
17703206
Type:
Article
Language:
en
ISSN:
1750-2799
Appears in Collections:
All Paterson Institute for Cancer Research

Full metadata record

DC FieldValue Language
dc.contributor.authorKiseleva, Elena-
dc.contributor.authorAllen, Terence D-
dc.contributor.authorRutherford, Sandra A-
dc.contributor.authorMurray, Stephen M-
dc.contributor.authorMorozova, Ksenia N-
dc.contributor.authorGardiner, Fiona-
dc.contributor.authorGoldberg, Martin W-
dc.contributor.authorDrummond, Sheona P-
dc.date.accessioned2009-06-15T11:37:04Z-
dc.date.available2009-06-15T11:37:04Z-
dc.date.issued2007-
dc.identifier.citationA protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM). 2007, 2 (8):1943-53 Nat Protocen
dc.identifier.issn1750-2799-
dc.identifier.pmid17703206-
dc.identifier.doi10.1038/nprot.2007.251-
dc.identifier.urihttp://hdl.handle.net/10541/70458-
dc.description.abstractThis protocol details methods for the isolation of yeast nuclei from budding yeast (Saccharomyces cerevisiae) and fission yeast (Schizosaccharomyces pombe), immuno-gold labeling of proteins and visualization by field emission scanning electron microscopy (FESEM). This involves the removal of the yeast cell wall and isolation of the nucleus from within, followed by subsequent processing for high-resolution microscopy. The nuclear isolation step can be performed in two ways: enzymatic treatment of yeast cells to rupture the cell wall and generate spheroplasts (cells that have partially lost their cell wall and their characteristic shape), followed by isolation of the nuclei by centrifugation or homogenization; and whole cell freezing followed by manual cell rupture and centrifugation. This protocol has been optimized for the visualization of the yeast nuclear envelope (NE), nuclear pore complexes (NPCs) and associated cyto-skeletal structures. Samples once processed for FESEM can be stored under vacuum for weeks, allowing considerable time for image acquisition.en
dc.language.isoenen
dc.subject.meshCell Culture Techniques-
dc.subject.meshCell Fractionation-
dc.subject.meshCell Nucleus-
dc.subject.meshCytoskeleton-
dc.subject.meshImmunohistochemistry-
dc.subject.meshMicroscopy, Electron, Scanning-
dc.subject.meshNuclear Envelope-
dc.subject.meshNuclear Pore-
dc.subject.meshSaccharomyces cerevisiae-
dc.subject.meshSchizosaccharomyces-
dc.titleA protocol for isolation and visualization of yeast nuclei by scanning electron microscopy (SEM).en
dc.typeArticleen
dc.contributor.departmentInstitute of Cytology and Genetics, Russian Academy of Science, Novosibirsk, Russia. elka@bionet.nsu.ruen
dc.identifier.journalNature Protocolsen

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