Anti-Histone H4 （tri methyl K20） antibody - ChIP Grade （ab9053）

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Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade抗組蛋白H4產品信息：

Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade

• Product name：Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade
• Description

  Rabbit polyclonal to Histone H4 (tri methyl K20) - ChIP Grade

• SpecificityHistone H4 tri-methylated at Lys 20.
• Tested applicationsChIP/Chip, IHC-P, Electron Microscopy, IHC (PFA fixed), IHC-Fr, ICC/IF, Flow Cyt, WB, ChIP
• Species reactivity

  Reacts with: Mouse, Rat, Cow, Human, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Toxoplasma gondii

• Immunogen

  Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H4, tri methylated at K20.

  Read Abcam's proprietary immunogen policy

  (Peptide available as ab17567.)

• Positive control
  • Calf Thymus Histone Preparation; Hela whole cell extract This antibody gave a positive result in IHC in the following FFPE tissue: Human normal skin
• General notes

  In immunofluorescence: Human cells: The antibody stains a subset of cellular chromatin that is characteristically DAPI-rich (condensed, heterochromatic). The perinucleolar heterochromatin is particularly rich in trimethylated lysine 20 staining. Mouse: Prominent staining of a subset of centromeric or pericentromeric heterochromatin. Interphase: The cells within the culture show a considerable variability in the intensity of staining with the antibody. The relationship between trimethylation levels and cell cycle have not yet been determined but may be a contributor to the amount of methylation detected in each cell. Heterochromatic regions of the interphase nucleus are the primary sites of trimethylation observed by indirect immunofluroescence. Mitosis: Discrete chromosomal regions are labelled intensely, with lower level fluorescence throughout the remainder of the chromosome arms. Immunofluorescence staining was performed as part of the nuclear antibody characterisation program at www.cellnucleus.com. Trimenthylation at Lys20 of Histone H4 isa common hallmark of human cancer (Fraga et al. 2005).

Properties

• FormLiquid
• Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
• Storage bufferPreservative: 0.02% Sodium Azide
  Constituents: 1% BSA, PBS, pH 7.4
• Concentration Batch dependent within range: 100 µg at 0.6 - 1 mg/ml

  Find concentration of your lot

• PurityImmunogen affinity purified
• Purification notesPurified using Sulpholink column with specific peptide linked via its cysteine residue
• Primary antibody notes In immunofluorescence: Human cells: The antibody stains a subset of cellular chromatin that is characteristically DAPI-rich (condensed, heterochromatic). The perinucleolar heterochromatin is particularly rich in trimethylated lysine 20 staining. Mouse: Prominent staining of a subset of centromeric or pericentromeric heterochromatin. Interphase: The cells within the culture show a considerable variability in the intensity of staining with the antibody. The relationship between trimethylation levels and cell cycle have not yet been determined but may be a contributor to the amount of methylation detected in each cell. Heterochromatic regions of the interphase nucleus are the primary sites of trimethylation observed by indirect immunofluroescence. Mitosis: Discrete chromosomal regions are labelled intensely, with lower level fluorescence throughout the remainder of the chromosome arms. Immunofluorescence staining was performed as part of the nuclear antibody characterisation program at www.cellnucleus.com. Trimenthylation at Lys20 of Histone H4 isa common hallmark of human cancer (Fraga et al. 2005).
• Clonality Polyclonal
• IsotypeIgG
• Research Areas
  • Epigenetics and Nuclear Signaling
  • Histones
  • H4
  • Methylated
  • Epigenetics and Nuclear Signaling
  • ChIP'ing antibodies
  • ChIP'ing antibodies

Applications

Our Abpromise guarantee covers the use of ab9053 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Target

• FunctionCore component of nucleosome. Nucleosomes wrap and compact DNA into chromatin, limiting DNA accessibility to the cellular machineries which require DNA as a template. Histones thereby play a central role in transcription regulation, DNA repair, DNA replication and chromosomal stability. DNA accessibility is regulated via a complex set of post-translational modifications of histones, also called histone code, and nucleosome remodeling.
• Sequence similaritiesBelongs to the histone H4 family.
• Post-translational
  modificationsAcetylation at Lys-6 (H4K5ac), Lys-9 (H4K8ac), Lys-13 (H4K12ac) and Lys-17 (H4K16ac) occurs in coding regions of the genome but not in heterochromatin.
  Citrullination at Arg-4 (H4R3ci) by PADI4 impairs methylation.
  Monomethylation and asymmetric dimethylation at Arg-4 (H4R3me1 and H4R3me2a, respectively) by PRMT1 favors acetylation at Lys-9 (H4K8ac) and Lys-13 (H4K12ac). Demethylation is performed by JMJD6. Symmetric dimethylation on Arg-4 (H4R3me2s) by the PRDM1/PRMT5 complex may play a crucial role in the germ-cell lineage.
  Monomethylated, dimethylated or trimethylated at Lys-21 (H4K20me1, H4K20me2, H4K20me3). Monomethylation is performed by SET8. Trimethylation is performed by SUV420H1 and SUV420H2 and induces gene silencing.
  Phosphorylated by PAK2 at Ser-48 (H4S47ph). This phosphorylation increases the association of H3.3-H4 with the histone chaperone HIRA, thus promoting nucleosome assembly of H3.3-H4 and inhibiting nucleosome assembly of H3.1-H4.
  Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins. Monoubiquitinated at Lys-92 of histone H4 (H4K91ub1) in response to DNA damage. The exact role of H4K91ub1 in DNA damage response is still unclear but it may function as a licensing signal for additional histone H4 post-translational modifications such as H4 Lys-21 methylation (H4K20me).
  Sumoylated, which is associated with transcriptional repression.
  Crotonylation (Kcr) is specifically present in male germ cells and marks testis-specific genes in post-meiotic cells, including X-linked genes that escape sex chromosome inactivation in haploid cells. Crotonylation marks active promoters and enhancers and confers resistance to transcriptional repressors. It is also associated with post-meiotically activated genes on autosomes.
• Cellular localizationNucleus. Chromosome.

• Database links

  • Entrez Gene: 318846 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 3771854 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 3771893 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 3771908 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 3771935 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 3771938 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 3771941 Fruit fly (Drosophila melanogaster)
  • Entrez Gene: 3771947 Fruit fly (Drosophila melanogaster)

• Alternative names

  • H4_HUMAN antibody
  • H4F2 antibody
  • H4FN antibody

Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade images

• 

  - Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)

  Left panel: a fission yeast whole cell extract and recombinant (r) fission yeast histone H4.

  Right panel: fission yeast whole cell extracts from strains with only a single wt (h4) or mutant (h4K20R) gene were immunoblotted.

  Review by Steven Sanders submitted 13 August 2004

• 

  ChIP - Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)

  Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 6 µl of  ab9053 (blue), and 20 µl of protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.

• Western blot - Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)

  All lanes : Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053) at 1 µg/ml
  
  Lane 1 : Calf thymus histone lysate
  Lane 2 : Calf thymus histone lysate with Human Histone H4 (unmodified ) peptide (ab2622) at 1 µg/ml
  Lane 3 : Calf thymus histone lysate with Human Histone H4 (mono methyl K20) peptide (ab17043) at 1 µg/ml
  Lane 4 : Calf thymus histone lysate with Human Histone H4 (di methyl K20) peptide (ab14964) at 1 µg/ml
  Lane 5 : Calf thymus histone lysate with Human Histone H4 (tri methyl K20) peptide (ab17567) at 1 µg/ml
  Lane 6 : Calf thymus histone lysate with Human Histone H3 (tri methyl K4) peptide (ab1342) at 1 µg/ml
  Lane 7 : Calf thymus histone lysate with Human Histone H3 (tri methyl K9) peptide (ab1773) at 1 µg/ml
  
  Secondary
  Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution
  
  Predicted band size : 11 kDa
  Observed band size : 13 kDa (why is the actual band size different from the predicted?)

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)

  IHC image of Histone H4 (tri methyl K20) staining in Human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab9053, 0.2µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX.

  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

• 

  Immunofluorescence - Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)Michael Hendzel, Cross Cancer Inst, Edmonton, Canada

  HeLa cells immunofluorescently stained with anti-Tri Methyl Lysine 20 of histone H4 (ab9053). HeLa cells were cultured on glass coverslips, fixed with 4% paraformaldehyde, and immunofluorescently labeled with anti-Tri K20 (green) at 1/500 and DAPI (red).

  The image was collected using a 1.3 N.A. 40 X PlanApo Objective (Zeiss) using a Zeiss Axiovert 200M inverted fluorescent microscope and a SensiCam HQ (Cooke Corp.) 12-bit CCD. The images were collected using Universal Imaging MetaMorph v. 5.1 for Windows. A Physik Instruments piezo z-drive was used to collect images at 200 nm intervals throughout the depth of the cell nucleus. The digital images were processed to remove fluorescent background and colour composites were made from single focal planes using MetaMorph

• 

  Immunofluorescence - Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)Michael Henzel, Cross Cancer Inst, Edmonton, Canada

  A mouse 10T1/2 embryonic fibroblast immunofluorescently stained with anti Tri Methyl Lysine 20 of histone H4. Murine 10T1/2 embryonic fibroblasts were cultured on glass coverslips, fixed with paraformaldehyde and then immunofluorescently labeled using an anti-Tri Methyl K20 of histone H4 antibody at 1/500 (green). The nuclear chromatin was simultaneously visualized using DAPI (red).

  The image was collected using a 1.4 N.A. 100 X PlanApo Objective (Zeiss) using a Zeiss Axiovert 200M inverted fluorescent microscope and a Photometrics Cascade 16-bit CCD. The images were collected using Universal Imaging MetaMorph v. 5.1 for Windows. A Physik Instruments piezo z-drive was used to collect images at 200 nm intervals throughout the depth of the cell nucleus. The digital images were processed to remove fluorescent background and colour composites were made from single focal planes using MetaMorph.

• 

  Immunofluorescence - Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053)Michael Henzel, Cross Cancer Inst, Edmonton, Canada

  A field of HeLa cells stained with anti-Trimethy Lysine 20 of Histone H4.

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