Abstract
Gaining membrane vesicles from different plant species and tissue types is crucial for membrane studies. Membrane vesicles can be used for further purification of individual membrane types, and, for example, in studies of membrane enzyme activities, transport assays, and in proteomic analysis. Membrane isolation from some species, such as conifers, has proved to be more difficult than that of angiosperm species. In this paper, we describe steps for isolating cellular membranes from developing xylem, phloem, and lignin-forming tissue-cultured cells of Norway spruce, followed by partial enrichment of plasma membranes by aqueous polymer two-phase partitioning and purity analyses. The methods used are partially similar to the ones used for mono- and dicotyledonous plants, but some steps require discreet optimization, probably due to a high content of phenolic compounds present in the tissues and cultured cells of Norway spruce.
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Abbreviations
- BSA:
-
Bovine serum albumin
- DTT:
-
Dithiothreitol
- PEG:
-
Polyethylene glycol
- PVP:
-
Polyvinylpyrrolidone
- PVPP:
-
Insoluble polyvinylpolypyrrolidone
- SB:
-
Suspension and storage buffer
- UDP-Glc:
-
UDP-glucose
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Acknowledgments
We thank S. Holmström, E. Oja, and S. Salonvaara for assistance during the optimization of the protocols for Norway spruce. For funding we thank Viikki Doctoral Programme in Molecular Biosciences (E.V.), Integrative Life Science Doctoral Program (E.V.), Academy of Finland (A.K.; grant 251390), and DAAD (S.L.; grant D/07/09909).
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Väisänen, E. et al. (2018). Isolation and Purity Assessment of Membranes from Norway Spruce. In: Mock, HP., Matros, A., Witzel, K. (eds) Plant Membrane Proteomics. Methods in Molecular Biology, vol 1696. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7411-5_2
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DOI: https://doi.org/10.1007/978-1-4939-7411-5_2
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