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Isolation of plastids and mitochondria from Chromera velia
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SYSNO ASEP 0520514 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title Isolation of plastids and mitochondria from Chromera velia Author(s) Sharaf, Abdoallah (BC-A) RID, ORCID
Füssy, Zoltán (BC-A) RID, ORCID
Tomčala, Aleš (BC-A) RID
Richtová, Jitka (BC-A) SAI
Oborník, Miroslav (BC-A) RID, ORCIDNumber of authors 5 Source Title Planta. - : Springer - ISSN 0032-0935
Roč. 250, č. 5 (2019), s. 1731-1741Number of pages 11 s. Publication form Print - P Language eng - English Country DE - Germany Keywords phototrophic relatives ; life-cycle ; apicomplexan ; genome ; dinoflagellate ; morphology ; cells ; red ; ultrastructure ; chloroplasts ; Chromerids ; Isolation ; Microalgae ; Mitochondrion ; Plastid Subject RIV EF - Botanics OECD category Plant sciences, botany R&D Projects GA15-17643S GA ČR - Czech Science Foundation (CSF) GA16-24027S GA ČR - Czech Science Foundation (CSF) EF16_019/0000759 GA MŠMT - Ministry of Education, Youth and Sports (MEYS) Method of publishing Limited access Institutional support BC-A - RVO:60077344 UT WOS 000491965700024 EID SCOPUS 85070799974 DOI 10.1007/s00425-019-03259-3 Annotation Main conclusionWe present an easy and effective procedure to purify plastids and mitochondria from Chromera velia. Our method enables downstream analyses of protein and metabolite content of the organelles.AbstractChromerids are alveolate algae that are the closest known phototrophic relatives to apicomplexan parasites such as Plasmodium or Toxoplasma. While genomic and transcriptomic resources for chromerids are in place, tools and experimental conditions for proteomic studies have not been developed yet. Here we describe a rapid and efficient protocol for simultaneous isolation of plastids and mitochondria from the chromerid alga Chromera velia. This procedure involves enzymatic treatment and breakage of cells, followed by differential centrifugation. While plastids sediment in the first centrifugation step, mitochondria remain in the supernatant. Subsequently, plastids can be purified from the crude pellet by centrifugation on a discontinuous 60%/70% sucrose density gradient, while mitochondria can be obtained by centrifugation on a discontinuous 33%/80% Percoll density gradient. Isolated plastids are autofluorescent, and their multi-membrane structure was confirmed by transmission electron microscopy. Fluorescent optical microscopy was used to identify isolated mitochondria stained with MitoTracker(TM) green, while their intactness and membrane potential were confirmed by staining with MitoTracker(TM) orange CMTMRos. Total proteins were extracted from isolated organellar fractions, and the purity of isolated organelles was confirmed using immunoblotting. Antibodies against the beta subunit of the mitochondrial ATP synthase and the plastid protochlorophyllide oxidoreductase did not cross-react on immunoblots, suggesting that each organellar fraction is free of the residues of the other. The presented protocol represents an essential step for further proteomic, organellar, and cell biological studies of C. velia and can be employed, with minor optimizations, in other thick-walled unicellular algae. Workplace Biology Centre (since 2006) Contact Dana Hypšová, eje@eje.cz, Tel.: 387 775 214 Year of Publishing 2020 Electronic address https://link.springer.com/content/pdf/10.1007/s00425-019-03259-3.pdf
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