Premium
Microscale magnetic microparticle‐based immunopurification of cytokinins from Arabidopsis root apex
Author(s) -
Plačková Lenka,
Oklestkova Jana,
Pospíšková Kristýna,
Poláková Kateřina,
Buček Jan,
Stýskala Jakub,
Zatloukal Marek,
Šafařík Ivo,
Zbořil Radek,
Strnad Miroslav,
Doležal Karel,
Novák Ondřej
Publication year - 2017
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1111/tpj.13443
Subject(s) - microscale chemistry , meristem , arabidopsis , biology , arabidopsis thaliana , chromatography , plant biology , biophysics , microbiology and biotechnology , biochemistry , botany , chemistry , mutant , shoot , mathematics education , mathematics , gene
Summary Cytokinins ( CK s) 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 CK s 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 CK s. 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 CK s (bases, ribosides and N 9 ‐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.