TOP2 | DNA topoisomerase II
AS05 096 | clonality: polyclonal | host: rabbit | reactivity: Arabidopsis thaliana, Vicia faba
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1: 2000 (WB), 1: 500 (IL)
|Expected | apparent MW||
164 | 170 kDa (Arabidopsis thaliana)
Arabidopsis thaliana, Vicia faba
Brassica rapa, Chlamydomonas reinhardtii, Chlorella vulgaris, Citrus clementina, Glycine max, Hordeum vulgare, Medicago truncatula, Oryza sativa, Ostreococcus tauri, Panicum italicum, Phaseolus vulgaris, Physcomitrella patens, Pinus sitchensis, Populus trichocarpa, Solanum tuberosum, Sorghum bicolor, Triticum aestivum, Vitis vinifera, Volvox caterii
|Not reactive in||
Topoisomerase II is highly expressed in young seedlings. The protein is localized in the nucleus and gene expression levels are increased in proliferative tissues like shoot apex or root tip.
Xie S & Lam E (1994) Abundance of nuclear DNA topoisomerase II is correlated with proliferation in Arabidopsis thaliana. NAR 25:5729.
10 µg of total protein from (Cyt) Arabidopsis thaliana cytoplasmic fraction, (Nuc) Arabidopsis thaliana nuclear fraction were separated on SDS-PAGE and blotted 1h to nitrocellulose. Filters were probed with anti-TOP2 antibodies (AS05 096, 1:2000). Signal was detected with SuperSignal West Pico ECL (Pierce).
Seeds of field bean (Vicia faba L. subsp. minor var. Nadwiślański; DANKO Group; Sobiejuchy) were sterilized using sodium hypochlorite (0.3% v/v) and germinated in Petri dishes on wetted filter paper at room temperature. At 4 d after imbibition, dark-grown seedlings with primary roots 25±5 mm long were selected for experiments. During incubations roots were oriented horizontally in a humid chamber and aerated continuously on a rotary water-bath shaker (30 rpm) at 23°C. Immunocytochemical assays were performed according to the method prescribed earlier (Rybaczek and Maszewski 2006). Excised apical parts of roots (1.5 mm long) were fixed for 45 min (18°C) in PBS-buffered 3.7% paraformaldehyde, washed several times with PBS and placed in a citric acid-buffered digestion solution (pH 5.0; 37°C for 45 min) containing 2.5% pectinase (Fluka), 2.5% cellulase (Onozuka R-10; Serva) and 2.5% pectoliase (ICN). After removing the digestion solution, root tips were washed 3 times in PBS, rinsed with distilled water and squashed onto Super Frost Plus glass slides (Menzel-Gläser). Air-dried slides were pretreated with PBS-buffered 5% BSA at 20°C for 50 min and incubated overnight in a humidified atmosphere (4°C) with rabbit antibody raised against TOPO2 (Agrisera), dissolved in PBS containing 1% BSA (at a dilution of 1:500). Following incubation, slides were washed 3 times with PBS and incubated for 1 h (18°C) with secondary goat anti-rabbit IgG DyLight®488 antibody (Agrisera, AS09 633, 1:3000). Nuclear DNA was stained with 4’,6-diamidino-2-phenyl-indole (DAPI, 0.4 μg/ml; Sigma-Aldrich). Following washing with PBS, slides were air dried and embedded in Vectashield Mounting Media for Fluorescence (Vector Laboratories). Observations were made using Optiphot-2 fluorescence microscope (Nikon) equipped with B-2A filter (blue light; λ ≈ 495 nm) for DyLight-conjugated antibodies and UV-2A filter (UV light; λ ≈ 365 nm) for DAPI. All images were recorded at exactly the same time of integration using DXM 1200 CCD camera.
Courtesy Dr. Dorota Rybaczek, Lodz University, Poland
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