Počet záznamů: 1
Isolation of plastids and mitochondria from Chromera velia
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SYSNO ASEP 0520514 Druh ASEP J - Článek v odborném periodiku Zařazení RIV J - Článek v odborném periodiku Poddruh J Článek ve WOS Název Isolation of plastids and mitochondria from Chromera velia Tvůrce(i) 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, ORCIDCelkový počet autorů 5 Zdroj.dok. Planta. - : Springer - ISSN 0032-0935
Roč. 250, č. 5 (2019), s. 1731-1741Poč.str. 11 s. Forma vydání Tištěná - P Jazyk dok. eng - angličtina Země vyd. DE - Německo Klíč. slova phototrophic relatives ; life-cycle ; apicomplexan ; genome ; dinoflagellate ; morphology ; cells ; red ; ultrastructure ; chloroplasts ; Chromerids ; Isolation ; Microalgae ; Mitochondrion ; Plastid Vědní obor RIV EF - Botanika Obor OECD Plant sciences, botany CEP GA15-17643S GA ČR - Grantová agentura ČR GA16-24027S GA ČR - Grantová agentura ČR EF16_019/0000759 GA MŠMT - Ministerstvo školství, mládeže a tělovýchovy Způsob publikování Omezený přístup Institucionální podpora BC-A - RVO:60077344 UT WOS 000491965700024 EID SCOPUS 85070799974 DOI 10.1007/s00425-019-03259-3 Anotace 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. Pracoviště Biologické centrum (od r. 2006) Kontakt Dana Hypšová, eje@eje.cz, Tel.: 387 775 214 Rok sběru 2020 Elektronická adresa https://link.springer.com/content/pdf/10.1007/s00425-019-03259-3.pdf
Počet záznamů: 1