Protocol abstract
Nature Protocols 2, - 259 - 262 (2007)
Published online: 22 February 2007 | doi:10.1038/nprot.2007.26
Subject Categories: Isolation, purification and separation | Model organisms | Cell and developmental biology | Plant biology
Isolation of intact vacuoles from Arabidopsis rosette leaf–derived protoplasts
Stéphanie Robert1, Jan Zouhar1,2, Clay Carter1,3 & Natasha Raikhel1
Abstract
Vacuoles are very prominent compartments within plant cells, and understanding of their function relies on knowledge of their content. Here, we present a simple vacuole purification protocol that was successfully used for large-scale isolation of vacuoles, free of significant contamination from other endomembrane compartments. This method is based on osmotic and thermal disruption of mesophyl-derived Arabidopsis protoplasts, followed by a density gradient fractionation of the cellular content. The whole procedure, including protoplast isolation, takes approximately 6 h.
- Department of Botany and Plant Sciences, Institute for Integrative Genome Biology, Center for Plant Cell Biology, University of California, Riverside, California 92521, USA.
- Present addresses. Departamento de Genética Molecular de Plantas, Centro Nacional de Biotecnología, Consejo Superior de Investigaciones Científicas, Madrid E-28049, Spain.
- Department of Biology, University of Minnesota, Duluth, Minnesota 55812, USA.
Correspondence to: Natasha Raikhel1 e-mail: nraikhel@ucr.edu
nature-products
A-Z product listing
- 150 ml Pyrex brand fleaker beaker(Fisher)
- 2-(N-morpholino)ethanesulfonic acid(VWR)
- 250 ml Kimax flasks with side arm(Fisher)
- 50-ml BD Falcon centrifuge tubes, polypropylene, conical bottom, sterile(VWR)
- Allegra 6 benchtop centrifuge(Beckman Coulter)
- Axioskop 40 Pol upright microscope(Zeiss)