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A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction
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SYSNO ASEP 0385990 Document Type J - Journal Article R&D Document Type Journal Article Subsidiary J Článek ve WOS Title A new approach for cytokinin isolation from Arabidopsis tissues using miniaturized purification: pipette tip solid-phase extraction Author(s) Svačinová, Jana (UEB-Q)
Novák, Ondřej (UEB-Q) RID, ORCID, SAI
Plačková, Lenka (UEB-Q) ORCID, RID
Lenobel, René (UEB-Q) ORCID
Holík, Josef (UEB-Q) RID, ORCID
Strnad, Miroslav (UEB-Q) RID, ORCID
Doležal, Karel (UEB-Q) RID, ORCIDSource Title Plant Methods. - : BioMed Central
Roč. 8, _ (2012), s. 17Number of pages 14 s. Language eng - English Country GB - United Kingdom Keywords Pipette tip solid-phase extraction (PT-SPE) ; Arabidopsis thaliana ; Cytokinins Subject RIV EC - Immunology R&D Projects TA01010861 GA TA ČR - Technology Agency of the Czech Republic (TA ČR) KAN200380801 GA AV ČR - Academy of Sciences of the Czech Republic (AV ČR) CEZ AV0Z50380511 - UEB-Q (2005-2011) UT WOS 000311423700001 DOI 10.1186/1746-4811-8-17 Annotation Background: We have developed a new analytical approach for isolation and quantification of cytokinins (CK) in minute amounts of fresh plant material, which combines a simple one-step purification with ultra-high performance liquid chromatography-fast scanning tandem mass spectrometry. Results: Plant tissue samples (1-5 mg FW) were purified by stop-and-go-microextraction (StageTip purification), which previously has only been applied for clean-up and pre-concentration of peptides. We found that a combination of two reverse phases and one cation-exchange phase, was the best tool, giving a total extraction recovery higher than 80%. The process was completed by a single chromatographic analysis of a wide range of naturally occurring cytokinins (bases, ribosides, O- and N-glucosides, and nucleotides) in 24.5 minutes using an analytical column packed with sub-2- microne particles. In multiple reaction monitoring mode, the detection limits ranged from 0.05 to 5 fmol and the linear ranges for most cytokinins were at least five orders of magnitude. The StageTip purification was validated and optimized using samples of Arabidopsis thaliana seedlings, roots and shoots where eighteen cytokinins were successfully determined. Conclusions: The combination of microextraction with one-step high-throughput purification provides fast, effective and cheap sample preparation prior to qualitative and quantitative measurements. Our procedure can be used after modification also for other phytohormones, depending on selectivity, affinity and capacity of the selected sorbents. Workplace Institute of Experimental Botany Contact David Klier, knihovna@ueb.cas.cz, Tel.: 220 390 469 Year of Publishing 2013
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