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

Overview

  • Product nameAnti-Histone H4 (tri methyl K20) antibody - ChIP GradeSee all Histone H4 primary antibodies ...
  • 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 more details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Toxoplasma gondii
    Predicted to work with: all Mammals
  • Immunogen

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

    (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 instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
  • Storage bufferPreservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • 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

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.

Application Notes
ChIP/Chip Use at an assay dependent concentration. PubMed: 19077033
IHC-P Use at an assay dependent dilution. PubMed: 20007528
Electron Microscopy Use at an assay dependent dilution. PubMed: 20543957
IHC (PFA fixed) Use at an assay dependent dilution. PubMed: 17083276
IHC-Fr Use at an assay dependent dilution. PubMed: 17712411
ICC/IF Use at an assay dependent dilution. PubMed: 19727073
Flow Cyt 1/100.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 13 kDa (predicted molecular weight: 11 kDa).Can be blocked with Human Histone H4 (tri methyl K20) peptide (ab17567).
ChIP Use 4-5µg for 106 cells.

Target

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

  • ab9053 staining Histone H4 (tri methyl K20) in human differentiated haematopoietic stem cells by Flow Cytometry. Cells were fixed with paraformaldehyde and permeabilized with permeabilization buffer. The sample was incubated with the primary antibody (1/300) for 12 hours at 4°C. An Alexa Fluor® 488-conjugated goat polyclonal anti-rabbit IgG (1/1000) was used as the secondary antibody.
    Gating Strategy: Isotype negative control (white).

     

    See Abreview

  • 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

  • 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.


  • Predicted band size : 11 kDa

    ab9053 is specific for Histone H4 (tri-methyl K20). This is illustrated in lane 5 where the activity of ab9053 is specifically blocked by the addition of the immunizing peptide (ab17567).



  • Predicted band size : 11 kDa

    ab9053 is specific for Histone H4 (tri-methyl K20). This is illustrated in lane 5 where the activity of ab9053 is specifically blocked by the addition of the immunizing peptide (ab17567).

  • 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.

  • 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).

    This image is part of the nuclear antibody characterisation program at www.cellnucleus.com

    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

  • 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).

    This image is part of the nuclear antibody characterisation program at www.cellnucleus.com

    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 com

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

    This image is part of the nuclear antibody characterisation program at www.cellnucleus.com

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

This product has been referenced in:
  • Thanisch K  et al. Targeting and tracing of specific DNA sequences with dTALEs in living cells. Nucleic Acids Res 42:e38 (2014). ICC/IF ; Human . Read more (PubMed: 24371265) »
  • Harding JL  et al. Small RNA profiling of Xenopus embryos reveals novel miRNAs and a new class of small RNAs derived from intronic transposable elements. Genome Res 24:96-106 (2014). ChIP ; Xenopus laevis, Xenopus tropicalis . Read more (PubMed: 24065776) »

See all 55 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 10 minute(s) · Concentration: 1% · Temperature: 21°C
Sample Human Cell (HEK293)
Specification HEK293
Permeabilization Yes - triton
Fixative Methanol
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Submitted Apr 23 2014

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - eBioscience Permeabilization Buffer
Sample Human Cell (Differentiated Haematopoietic Stem Cells)
Specification Differentiated Haematopoietic Stem Cells
Gating Strategy Isotype negative control (white)
Preparation Cell harvesting/tissue preparation method: Typsin-EDTA Cell Dissociation
Sample buffer: PBS and 10% FBS
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Submitted Feb 20 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Human Tissue lysate - whole (Patient Dermal Fibroblasts)
Specification Patient Dermal Fibroblasts
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Feb 01 2014

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing
Sample Human Cell lysate - whole cell (Human Embryonic kidney cell lines)
Specification Human Embryonic kidney cell lines
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
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Submitted Nov 25 2013

Application Western blot
Loading amount 4.5 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Fruit fly (Drosophila melanogaster) Tissue lysate - nuclear (Fruit fly embryo)
Specification Fruit fly embryo
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Nov 12 2013

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Human Cell lysate - whole cell (Human HeLa cells)
Specification Human HeLa cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Nov 12 2013

Application Western blot
Loading amount 8 µg
Gel Running Conditions Reduced Denaturing (12%)
Sample Caenorhabditis elegans Tissue lysate - nuclear (C. elegans whole body)
Specification C. elegans whole body
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Nov 12 2013

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Application Western blot
Sample Mouse Tissue lysate - whole (Striatum (Brain))
Loading amount 50 µg
Specification Striatum (Brain)
Gel Running Conditions Reduced Denaturing (12%)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
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Submitted Nov 07 2012

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"