Dehydrin (affinity purified serum)
AS07 206A | Clonality: Polyclonal | Host: Rabbit | Reactivity: Higher plants
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KLH-conjugated peptide sequence TGEKKGIMDKIKEKLPGQH of K-segment conserved in a wide range of plant species
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Line C2- 10 µg of Brassica oleracea mitochondrial proteins (C2- plants grown in severe drought conditions) isolated as described by Rurek et al., 2015 (doi: 10.1016/j.bbabio.2015.01.005) were separated by 12% SDS-PAGE and electroblotted in semi-dry conditions (Towbin buffer) to Immobilon-P membrane (Millipore). Blots were CBB R 250 briefly stained, destained, wet-scanned and after completed destaining, they were blocked in 5% skimmed milk (dissolved in PBS-T containing 0.1% Tween 20) in 1h, RT. Primary antisera (at 1: 1000, diluted in 2% skimmed milk in PBS-T) were bound by overnight incubation of blots at 4°C. After blot washing (2 times quick, 2 times of 5 min, and 10 min at the end), secondary goat anti-rabbit IgGs, HRP- conjugated (Agrisera product AS09 602; at 1: 50000, diluted in 2% milk/ PBS-T) were bound in 1 h, RT. Blots were washed (as above) with copious amounts of PBS-T and chemiluminescence signals acquired by using standard ECL reagents on RTG film between 3 s and 2 min (periods of the given image acquisition were indicated).
In the nuclear fraction of Arabidopsis thaliana, few HMW proteins immunoreactive with anti-dehydrin anstiserum were also detected. The differences between their abundance in WT and 9-11 lines could be, however, ascribed to the actual protein loading on the blot. Experimental procedure was conducted as above.
Lines WT and 9-11- ca. 20 µg of proteins enriched in nuclear fraction from Arabidopsis thaliana leaf rosettes (WT- wild type; 9-11- T-DNA insertion mutants for h2a.z gene encoding particular histon H2A.Z isoform) were resolved by SDS-PAGE and HMW (high-molecular weight) proteins immunoreactive with dehydrin antisera were analysed. After separation, proteins were electroblotted and immunodetected essentially in the same manner as it was indicated above.
Application of WT/9-11 blot was possible due to the courtesy of the Department of Biotechnology (Institute of Molecular Biology and Biotechnology, Faculty of Biology, Adam Mickiewicz University in Poznań). All remaining blots as well as all immunodetection assays were prepared by Dr Michał Rurek (Department of Molecular and Cellular Biology of the same Institute).
Dehydrins are stress proteins involved in formation of plant protective reactions against dehydration. They are normally synthesized in maturating seeds during their dessication, as well as in vegetative tissues of plants treated with abscisic acid or exposed to environmental stress factors that result in cellular dehydration.
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