H3 | Histone H3 (rabbit antibody) (nuclear marker)
AS10 710 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, C. reinhardtii, S. lycopersicum, V. faba, P. patens, S. europaea, Z. mays | cellular [compartment marker] of nucleoplasm
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|Recommended dilution||1 : 100-1 : 500 (ICC), 2 µl of antibody/500 µl solution (ChIp-qPCR), 1 : 5000 (WB)|
|Expected | apparent MW||
15 | 17 kDa
|Confirmed reactivity||Arabidopsis thaliana, Brassica oleracea, Capsicum annuum, Chlamydomonas acidophila, Chlamydomonas reinhardtii, Cucumis sativus L cv Suyo, Nicotiana benthamiana, Physcomitrella patens, Salicornia europaea, Solanum lycopersicum, Solanum sogarandinum, Solanum tuberosum, Vicia faba, Zea mays|
Brachypodium distachyon, Brassica napus, Emiliania huxleyi, Hordeum vulgare, Nicotiana tabacum, Malus domestica, Medicago sativa, Triticum aestivum, Pinus pinaster, Pisum sativum, Oryza sativa, Zea mays, Vitis vinifera, Volvox sp.
|Not reactive in||No confirmed exceptions from predicted reactivity are currently known.|
This product can be sold containing ProClin if requested
Protocol for isolation of cytosolic and nuclear fractions can be found here.
Specific fluorescence in ICC has been observed for interphase nuclei as well as around centromer region (where Ser10 of histone H3 is phosphorylated) in mitotic chromosomes.
|Selected references||Hartmann et al. (2018). Subcellular Compartmentation of Alternatively Spliced Transcripts Defines SERINE/ARGININE-RICH PROTEIN30 Expression. Plant Physiol. 2018 Apr;176(4):2886-2903. doi: 10.1104/pp.17.01260.
Duan et al. (2017). A Lipid-Anchored NAC Transcription Factor Is Translocated into the Nucleus and Activates Glyoxalase I Expression during Drought Stress. Plant Cell. 2017 Jul;29(7):1748-1772. doi: 10.1105/tpc.17.00044. (Nicotiana benthamiana)
Rihan et al. (2017). An analysis of the development of cauliflower seed as a model to improve the molecular mechanism of abiotic stress tolerance in cauliflower artificial seeds. Plant Physiol Biochem. 2017 Jul;116:91-105. doi: 10.1016/j.plaphy.2017.05.011.
Shin et al. (2017). The metabolic sensor AKIN10 modulates the Arabidopsis circadian clock in a light-dependent manner. Plant Cell Environ. 2017 Jan 5. doi: 10.1111/pce.12903.
Correa-Galvis et al. (2016). Photosystem II Subunit PsbS Is Involved in the Induction of LHCSR Protein-dependent Energy Dissipation in Chlamydomonas reinhardtii. J Biol Chem. 2016 Aug 12;291(33):17478-87. doi: 10.1074/jbc.M116.737312.
Castellano et al. (2016). A pathogenic long noncoding RNA redesigns the epigenetic landscape of the infected cells by subverting host Histone Deacetylase 6 activity. New Phytol. 2016 Sep;211(4):1311-22. doi: 10.1111/nph.14001. Epub 2016 May 12.
Ghandi et al. (2016). Tomato yellow leaf curl virus infection mitigates the heat stress response of plants grown at high temperature. Sci Rep. 2016 Jan 21;6:19715. doi: 10.1038/srep19715.
Gorovits et al. (2016). Tomato yellow leaf curl virus confronts host degradation by sheltering in small/midsized protein aggregates. Virus Res. 2016 Feb 2;213:304-13. doi: 10.1016/j.virusres.2015.11.020. Epub 2015 Dec 1
|1.2 μg of Arabidopsis thaliana chromatin-enriched fraction (1) and 3.75 µg of total protein from 4-weeks-old Arabidopsis thaliana leaves (2), and were separated on 12% SDS-PAGE and blotted 50 mins to Immobilon-P (Millipore, semi-dry) PVDF membrane. Blots were blocked immediately following transfer in MTBS-T (5% milk) for 30 mins at room temperature with agitation. Blots were incubated in the primary antibody at a dilution of 1:5000 for 1h at room temperature with agitation. The antibody solution was decanted and the blot was rinsed briefly twice, then washed 3 times for 3 min in TBS-T at room temperature with agitation. Blots were incubated in secondary antibody (anti- IgG horse radish peroxidase conjugated, from Agrisera, AS09 602) diluted to 1:20 000 for 30 mins at room temperature with agitation. The blots were washed as above and developed for 5 min with ECL detection reagent (Roche) according to the manufacturers instructions. Exposure time was 30 seconds. Double band in chromatine-enriched fraction (1) has been outcompeted in peptide neutralization assay by peptide used to elicit H3 antibodies. Chromatin izolation was carried out as described (Zilberman et al. 2008) with minor modifications.
Courtesy of Weronika Sura and Dr. Piotr A. Ziolkowski (Department of Biotechnology, Adam Mickiewicz University, Poznan, Poland)
|30 μg of 5 µl of Chlamydomonas reinhardtii protein saturated in 8M urea were separated on 15% SDS-PAGE and blotted for 1hour to 0.2 µm nitrocellulose at 100V using wet transfer system. Blots were blocked with 0.5% cold fish gelatin for 1hr at room temp with agitation. Blot was incubated in the primary antibody (anti-H3) at a dilution of 1:2500 for an hour at RT with agitation. The blots were washed with 3X 15min TBS-TT at RT with agitation. Blots as incubated in the secondary antibody (DayLight 800) 1:5000 dilution for 30min at RT with agitation and washed 1X with TBSTT for 15min, 1X with TBST for 15min before scanning with the ODyssey IRD scanner.
Courtesy of Dr. Betty Chung, University of Cambridge, United Kingdom
5 µl of 15μg/µl Solanum lycopersicum protein saturated in 8M urea were separated on 15% SDS-PAGE and blotted for 1hour to 0.2 µm nitrocellulose at 100V using wet transfer system. Blots were blocked with 0.5% cold fish gelatin for 1hr at room temp with agitation. Blot was incubated in the primary antibody (anti-H3) at a dilution of 1:2500 for an hour at RT with agitation. The blots were washed with 3X 15min TBS-TT at RT with agitation. Blots as incubated in the secondary antibody, fluorescent antibody (AS12 2460, Agrisera) 1:5000 dilution for 30min at RT with agitation and washed 1X with TBSTT for 15min, 1X with TBST for 15min before scanning with the ODyssey IRD scanner.
Courtesy of Dr. Betty Chung and Dr Zhengming Wang, University of Cambridge, United Kingdom
Immunocytochemical assays were performed according to the method described earlier (Rybaczek and Maszewski 2006). Excised apical parts of Vicia faba 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 H3 histone (Agrisera), dissolved in PBS containing 1% BSA (at a dilution of 1:50). Following incubation, slides were washed 3 times with PBS and incubated for 1 h (18°C) with Agrisera secondary goat anti-rabbit IgG DyLight®488 antibody (AS09 633, 1:1000). 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, Łódź University, Poland
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