title: Microscale magnetic microparticle-based immunopurification of cytokinins from Arabidopsis root apex.
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-1fresh 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.
authors.affiliation: Laboratory of Growth Regulators, Centre of Region Haná for Biotechnological and Agricultural Research, Faculty of Sciences of Palacký University and Institute of Experimental Botany, Czech Academy of Sciences, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.|Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science of Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Lenka
authors.initials: L
authors.lastname: Plačková
authors.affiliation: Laboratory of Growth Regulators, Centre of Region Haná for Biotechnological and Agricultural Research, Faculty of Sciences of Palacký University and Institute of Experimental Botany, Czech Academy of Sciences, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Jana
authors.initials: J
authors.lastname: Oklestkova
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Kristýna
authors.initials: K
authors.lastname: Pospíšková
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Kateřina
authors.initials: K
authors.lastname: Poláková
authors.affiliation: Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science of Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Jan
authors.initials: J
authors.lastname: Buček
authors.affiliation: Department of Organic Chemistry, Faculty of Science, Palacký University, 17 Listopadu 12, CZ-77146, Olomouc, Czech Republic.
authors.forename: Jakub
authors.initials: J
authors.lastname: Stýskala
authors.affiliation: Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science of Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Marek
authors.initials: M
authors.lastname: Zatloukal
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.|Department of Nanobiotechnology, Biology Centre, ISB, CAS, Na Sádkách 7, CZ-37005, České Budějovice, Czech Republic.
authors.forename: Ivo
authors.initials: I
authors.lastname: Šafařík
authors.affiliation: Regional Centre of Advanced Technologies and Materials, Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Radek
authors.initials: R
authors.lastname: Zbořil
authors.affiliation: Laboratory of Growth Regulators, Centre of Region Haná for Biotechnological and Agricultural Research, Faculty of Sciences of Palacký University and Institute of Experimental Botany, Czech Academy of Sciences, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Miroslav
authors.initials: M
authors.lastname: Strnad
authors.affiliation: Laboratory of Growth Regulators, Centre of Region Haná for Biotechnological and Agricultural Research, Faculty of Sciences of Palacký University and Institute of Experimental Botany, Czech Academy of Sciences, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.|Department of Chemical Biology and Genetics, Centre of the Region Haná for Biotechnological and Agricultural Research, Faculty of Science of Palacký University, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Karel
authors.initials: K
authors.lastname: Doležal
authors.affiliation: Laboratory of Growth Regulators, Centre of Region Haná for Biotechnological and Agricultural Research, Faculty of Sciences of Palacký University and Institute of Experimental Botany, Czech Academy of Sciences, Šlechtitelů 27, CZ-78371, Olomouc, Czech Republic.
authors.forename: Ondřej
authors.initials: O
authors.lastname: Novák
mesh_terms: D017360:Arabidopsis / Q000737:chemistry*; D003583:Cytokinins / Q000737:chemistry / Q000302:isolation & purification*; D058185:Magnetite Nanoparticles; D010937:Plant Growth Regulators / Q000737:chemistry / Q000302:isolation & purification*; D018517:Plant Roots / Q000737:chemistry*
chemical_list: D003583:Cytokinins; D058185:Magnetite Nanoparticles; D010937:Plant Growth Regulators
keywords: 
  Arabidopsis thaliana
  ; cytokinins; immunoaffinity separation; magnetic microparticles; metabolite profiling; technical advance