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Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex |
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| Authors: | Lenka Pla?ková Jana Oklestkova Kristýna Pospí?ková Kate?ina Poláková Jan Bu?ek Jakub Stýskala Marek Zatloukal Ivo ?afa?ík Radek Zbo?il Miroslav Strnad Karel Dole?al Ond?ej Novák |
| Affiliation: | 1. Laboratory of Growth Regulators, Centre of Region Haná for Biotechnological and Agricultural Research, Faculty of Sciences of Palacky University and Institute of Experimental Botany, Czech Academy of Sciences, Olomouc, Czech Republic;2. Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science of Palacky University, Olomouc, Czech Republic;3. Regional Centre of Advanced Technologies and Materials, Palacky University, Olomouc, Czech Republic;4. Department of Organic Chemistry, Faculty of Science, Palacky University, Olomouc, Czech Republic;5. Department of Nanobiotechnology, Biology Centre, ISB, CAS, ?eské Budějovice, Czech Republic |
| Abstract: | Cytokinins (CKs) are pivotal plant hormones that have crucial roles in plant growth and development. However, their isolation and quantification are usually challenging because of their extremely low levels in plant tissues (pmol g?1 fresh weight). We have developed a simple microscale magnetic immunoaffinity‐based method for selective one‐step isolation of CKs from very small amounts of plant tissue (less than 0.1 mg fresh weight). The capacity of the immunosorbent and the effect of the complex plant matrix on the yield of the rapid one‐step purification were tested using a wide range of CK concentrations. The total recovery range of the new microscale isolation procedure was found to be 30–80% depending on individual CKs. Immunoaffinity extraction using group‐specific monoclonal CK antibodies immobilized onto magnetic microparticles was combined with a highly sensitive ultrafast mass spectrometry‐based method with a detection limit close to one attomole. This combined approach allowed metabolic profiling of a wide range of naturally occurring CKs (bases, ribosides and N9‐glucosides) in 1.0‐mm sections of the Arabidopsis thaliana root meristematic zone. The magnetic immunoaffinity separation method was shown to be a simple and extremely fast procedure requiring minimal amounts of plant tissue. |
| Keywords: | cytokinins magnetic microparticles immunoaffinity separation metabolite profiling Arabidopsis thaliana technical advance |
