Methods for studying the yeast vacuole

Christopher J. Roberts, Christopher K. Raymond, Carl Yamashiro, Tom H. Stevens

Research output: Contribution to journalArticle

278 Citations (Scopus)

Abstract

This chapter describes techniques used in the study of the vacuole and focuses on the procedures used in the laboratories. The yeast vacuole is a large, dynamic organelle that can be detected by fight microscopy. It is most prominent when cells are viewed using differential interference-contrast optics. Reliable detection of vacuoles using vital staining with fluorescent dyes in conjunction with fluorescence microscopy is described in the chapter. Several methods are available for purification of the yeast vacuole, all of which take advantage of the low buoyant density of the vacuole. The method of choice is a modified version of the procedure used by involves enzymatic removal of the cell walls, osmotic lysis of the spheroplasts, followed by flotation of the vacuoles on a discontinuous Ficoll gradient. Other methods use polybase induced lysis of spheroplasts under isotonic conditions followed by density gradient centrifugation. The procedure that follows has consistently given 10–20% yield of vacuoles and low contamination of other organelles.

Original languageEnglish (US)
Pages (from-to)644-661
Number of pages18
JournalMethods in Enzymology
Volume194
Issue numberC
DOIs
StatePublished - Jan 1 1991
Externally publishedYes

Fingerprint

Vacuoles
Yeast
Yeasts
Ficoll
Centrifugation
Fluorescence microscopy
Flotation
Fluorescent Dyes
Purification
Optics
Microscopic examination
Contamination
Spheroplasts
Cells
Organelles
Density Gradient Centrifugation
Fluorescence Microscopy
Cell Wall
Microscopy
Staining and Labeling

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

Cite this

Methods for studying the yeast vacuole. / Roberts, Christopher J.; Raymond, Christopher K.; Yamashiro, Carl; Stevens, Tom H.

In: Methods in Enzymology, Vol. 194, No. C, 01.01.1991, p. 644-661.

Research output: Contribution to journalArticle

Roberts, CJ, Raymond, CK, Yamashiro, C & Stevens, TH 1991, 'Methods for studying the yeast vacuole', Methods in Enzymology, vol. 194, no. C, pp. 644-661. https://doi.org/10.1016/0076-6879(91)94047-G
Roberts, Christopher J. ; Raymond, Christopher K. ; Yamashiro, Carl ; Stevens, Tom H. / Methods for studying the yeast vacuole. In: Methods in Enzymology. 1991 ; Vol. 194, No. C. pp. 644-661.
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