H3 | Histone H3 (rabbit antibody) (nuclear marker)
AS10 710 | Clonality: Polyclonal | Host: Rabbit | Reactivity: A. thaliana, C. reinhardtii, H. vulgare, S. lycopersicum, V. faba, P. patens, S. europaea, Z. mays | cellular [compartment marker] of nucleoplasm
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KLH-conjugated synthetic peptide derived from known H3 sequences, inluding Arabidopsis thaliana H3.3 P59169 (At4g40030, At4g40040, At5g10980), H3.2 P59226 (At1g09200, At3g27360, At5g10390, At5g10400, At5g65360), H3-like 2 Q9FXI7 (At1g19890)
15 | 17 kDa
Brachypodium distachyon, Brassica napus, Emiliania huxleyi, Nannochloropsis gaditana, Nicotiana tabacum, Malus domestica, Medicago sativa, Triticum aestivum, Pinus pinaster, Pisum sativum, Oryza sativa, Ostreococcus sp. , Vitis vinifera, Volvox sp.
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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 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
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
Cellular [compartment marker] of nucleoplasm, loading control antibody for Chlamydomonas reinhardtii
Histone 3 (H3) located in nuclei, incorporated into chromatin. Present in nucleosome together with H2A, H2B and H4.
Perlaza et al. (2019). The Mars1 kinase confers photoprotection through signaling in the chloroplast unfolded protein response. Elife. 2019 Oct 15;8. pii: e49577. doi: 10.7554/eLife.49577. (immunofluorescence)
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Wang et al. (2018). Degradation of unmethylated miRNA/miRNA*s by a DEDDy-type 3' to 5' exoribonuclease Atrimmer 2 in Arabidopsis. Proc Natl Acad Sci U S A. 2018 Jul 10;115(28):E6659-E6667. doi: 10.1073/pnas.1721917115.
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)
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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.
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