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Isolation of Vacuoles from the Leaves of the Medicinal Plant Catharanthus roseus.
- Source :
-
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2018; Vol. 1789, pp. 81-99. - Publication Year :
- 2018
-
Abstract
- The isolation of vacuoles is an essential step to unravel the important and complex functions of this organelle in plant physiology. Here, we describe a method for the isolation of vacuoles from Catharanthus roseus leaves involving a simple procedure for the isolation of protoplasts, and the application of a controlled osmotic/thermal shock to the naked cells, leading to the release of intact vacuoles, which are subsequently purified by density gradient centrifugation. The purity of the isolated intact vacuoles is assayed by microscopy, western blotting, and measurement of vacuolar (V)-H <superscript>+</superscript> -ATPase hydrolytic activity. Finally, membrane functionality and integrity is evaluated by measuring the generation of a transtonoplast pH gradient by the V-H <superscript>+</superscript> -ATPase and the V-H <superscript>+</superscript> -pyrophosphatase, also producing further information on vacuole purity.
- Subjects :
- Benzenesulfonates analysis
Blotting, Western methods
Catharanthus metabolism
Enzyme Assays methods
Fluoresceins analysis
Fluorescent Dyes analysis
Hydrolysis
Microscopy, Fluorescence methods
Neutral Red analysis
Optical Imaging methods
Osmotic Pressure
Plant Leaves metabolism
Plant Proteins analysis
Plant Proteins metabolism
Plants, Medicinal cytology
Plants, Medicinal metabolism
Protoplasts cytology
Protoplasts metabolism
Protoplasts ultrastructure
Pyridinium Compounds analysis
Quaternary Ammonium Compounds analysis
Staining and Labeling methods
Vacuolar Proton-Translocating ATPases analysis
Vacuolar Proton-Translocating ATPases metabolism
Catharanthus cytology
Cell Fractionation methods
Plant Leaves cytology
Vacuoles metabolism
Vacuoles ultrastructure
Subjects
Details
- Language :
- English
- ISSN :
- 1940-6029
- Volume :
- 1789
- Database :
- MEDLINE
- Journal :
- Methods in molecular biology (Clifton, N.J.)
- Publication Type :
- Academic Journal
- Accession number :
- 29916073
- Full Text :
- https://doi.org/10.1007/978-1-4939-7856-4_7