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Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection

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Abstract

A rapid methodology for the simultaneous analysis of a large number of cytokinins is presented. The cross-reactivity of a mixture of polyclonal antibodies against zeatin riboside and isopentenyladenosine was exploited in a protocol that can be used for immunoaffinity purification of 23 additional cytokinins. Ligands include the cytokinin bases zeatin, dihydrozeatin, isopentenyladenine, benzyl-adenine and kinetin, and their corresponding nucleoside, nucleoside-5′-monophosphate, and 9-glucoside derivatives, as well as cis-zeatin, cis-zeatin riboside, the 2-methylthiol derivatives of isopentenyladenosine and zeatin riboside, and benzyl-adenine-3-glucoside. Mixtures of cytokinins could be retained with high recoveries of all the components. Immunoaffinity purification of extracts of Arabidopsis thaliana (L.) Heynh. and Solarium tuberosum L. gave fractions clean enough, as verified by gas chromatographymass spectrometry (GC-MS), to allow analysis of endogenous cytokinins using a single high-performance liquid chromatography (HPLC) step with on-line UV-spectrum detection. The detection limit was 4–6 pmol. The procedure described forms a routine assaying technique that is faster and simpler, yet yields better qualitative and quantitative information than the commonly used procedure of immunoassaying of HPLC fractions.

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Abbreviations

IAC:

immunoaffinity chromatography

ipA:

isopentenyladenosine, 9-β-d-ribofuranosyl-N 6-(Δ2-isopentenyl)-adenine

GC-MS:

gas chromatography-mass spectrometry

HPLC:

high-performance liquid chromatography

PBS:

Phosphate-buffered saline

ZR:

zeatin riboside 9-β-d-ribofuranosyl-zeatin

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Authors and Affiliations

  1. Department of Plant Physiology, Swedish University of Agricultural Science, P.O. Box 7047, S-75007, Uppsala, Sweden

    Björn Nicander, Ulf Ståhl, Per-Olof Björkman & Elisabeth Tillberg

Authors
  1. Björn Nicander
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  2. Ulf Ståhl
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  3. Per-Olof Björkman
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  4. Elisabeth Tillberg
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Additional information

This work was supported by a grant from the Swedish Natural Science Research Council. Björn Nicander was supported during part of the work by a grant from the Knut och Alice Wallenberg Foundation.

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Nicander, B., Ståhl, U., Björkman, PO. et al. Immunoaffinity co-purification of cytokinins and analysis by high-performance liquid chromatography with ultraviolet-spectrum detection. Planta 189, 312–320 (1993). https://doi.org/10.1007/BF00194427

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