H3 | Histone H3 (antigen affinity purified)

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)

Brassica oleracea, Capsicum annuum, Chlamydomonas acidophila, Chlamydomonas reinhardtii, Physcomitrium patens, Salicornia europaea, Solanum lycopersicum, Solanum sogarandinum, Solanum tuberosum, Vicia faba, Zea mays Brachypodium distachyon, Brassica napus, Hordeum vulgare, Nicotiana tabacum, Malus domestica, Medicago sativa, Nannochloropsis gaditana, Triticum aestivum, Pinus pinaster, Pisum sativum, Zea mays, Vitis vinifera, Volvox sp.

Application example

Immunofluorescent localization of Histone 3 on suspension culture of Arabidopsis thaliana (upper image) or Oryza sativa (bottom image), using anti-histone 3 antibodies (AS10 710A) and anti-rabbit IgG DyLight®488 conjugated secondary antibodies (AS10 1165). DAPI staining of nuclei is pseudocolored red. 

Material: Suspension cultures of Arabidopsis thaliana, ecotype Landsberg erecta cv.MM1 or Oryza sativa ssp.japonica cv. 'Unggi 9'
Fixation: Packed cell volume to fixer ratio: 250 µl : 5ml
Fixer composition and buffer: 4% (w/v) paraformaldehyde (freshly prepared as 8% stock and 0.2 µm filtered) in Phosphate Buffered Saline (PBS), pH 7.4 (2x stock, 0.2 µm filtered)
Container and method: in 6 cm Petri dish, gentle shaking at room temperature (RT)
Duration: 30 minutes (Arabidopsis thaliana) or 60 minutes (Oryza sativa). Cells were not shaken during the first 5 mins of fixation to allowed to partially recover from osmotic shock induced by formaldehyde.
Hydrophilization: no
Cell wall digestion: Yes
Packed cell volume to enzyme ratio: 100 µl : 2ml Enzyme composition: 1% (A) 1.2% (R) Cellulase (chromatically purified, powder, Worthington) 1% Pectinase (protease free, liquid, Sigma) Buffer: 0.5% (w/v) MES buffer, pH 5.6
Container and method: in 2 ml microfuge tube by rolling at room temperature (RT)
Duration: 30 minutes (Arabidopsis thaliana) or 90 minutes (Oryza sativa)
Membrane permeabilization: Triton-X100 (0.5%), 10 min/RT
Antigen retrieval: no
Blocking buffer: Fish gelatin (5% v/v)
Washing buffer: PBS
Primary antibody dilution and incubation time: 1:400, ON/4ºC
Secondary antibody dilution and incubation time and supplier: anti-rabbit IgG DyLight®488 conjugated secondary antibodies (AS10 1165), 1:600, 1hn/RT
Co-staining of the nucleus (DAPI): Yes
Nucleus staining: 100 ng/ml DAPI

Courtesy of Dr. Ferhan Ayaydin, Hungarian Centre of Excellence for Molecular Medicine (HCEMM), Szeged, Hungary. 

Protocol for isolation of cytosolic and nuclear fractions can be found here.

Specific protol for ChIP can be found here: Saleh et al. (2008).