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

Overview

  • Product nameAnti-Histone H4 (tri methyl K20) antibody - ChIP Grade
    See all Histone H4 primary antibodies
  • Description
    Rabbit polyclonal to Histone H4 (tri methyl K20) - ChIP Grade
  • SpecificityHistone H4 tri-methylated at Lys 20.
  • Tested applicationsSuitable for: ChIP/Chip, IHC-P, Electron Microscopy, IHC (PFA fixed), IHC-Fr, ICC/IF, Flow Cyt, WB, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Cow, Human, Schizosaccharomyces pombe, Toxoplasma gondii
    Predicted to work with: all Mammals
  • Immunogen

    Synthetic peptide within Human Histone H4 aa 1-100 (tri methyl K20) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
    (Peptide available as ab17567)

  • Positive control
    • WB: Calf thymus histone preparation and HeLa whole cell extract. IHC-P: Human normal skin tissue.
  • 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 notesIn 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).
  • ClonalityPolyclonal
  • 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 Abreviews Notes
ChIP/Chip Use at an assay dependent concentration. PubMed: 19077033
IHC-P Use at an assay dependent concentration. PubMed: 20007528
Electron Microscopy Use at an assay dependent concentration. PubMed: 20543957
IHC (PFA fixed) Use at an assay dependent concentration. PubMed: 17083276
IHC-Fr Use at an assay dependent concentration. PubMed: 17712411
ICC/IF Use at an assay dependent concentration. PubMed: 19727073
Flow Cyt 1/100.

ab171870 - Rabbit polyclonal IgG, is suitable for use as an isotype control with this antibody.

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

  • Immunohistochemical staining of paraffin embedded, PFA fixed, mouse liver using ab9053 at 1/100. Heat mediated antigen retrieval was performed using sodium citrate buffer, pH 6, and the sample was permeabilized with 0.05% Triton X 100. The sample was blocked in 5% BSA for 1 hour at room temperature and was then incubated with primary antibody for 3 hours in PBS/0.05% Triton X 100 at room temperature. An Alexa Fluor® 555 donkey anyti-rabbit was used as the secondary at 1/250.

    See Abreview

  • 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).

  • 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



  • Predicted band size : 11 kDa

    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

  • All lanes : Anti-Histone H4 (tri methyl K20) antibody - ChIP Grade (ab9053) at 1 µg/ml (Calf thymus histone lysate)

    Lane 1 : Human Histone H4 peptide (ab2622)
    Lane 2 : Human Histone H4 peptide (ab2622)
    Lane 3 : Human Histone H4 (mono methyl K20) peptide (ab17043)
    Lane 4 : Human Histone H4 (di methyl K20) peptide (ab14964)
    Lane 5 : Human Histone H4 (tri methyl K20) peptide (ab17567)
    Lane 6 : Human Histone H3 (tri methyl K4) peptide (ab1342)
    Lane 7 : Human Histone H3 (tri methyl K9) peptide (ab1773)

    Blocking peptides at 1 µg/ml per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution

    Predicted band size : 11 kDa

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

This product has been referenced in:
  • Khan SA  et al. p38-MAPK/MSK1-mediated overexpression of histone H3 serine 10 phosphorylation defines distance-dependent prognostic value of negative resection margin in gastric cancer. Clin Epigenetics 8:88 (2016). Read more (PubMed: 27588146) »
  • Svensson JP  et al. A nucleosome turnover map reveals that the stability of histone H4 Lys20 methylation depends on histone recycling in transcribed chromatin. Genome Res 25:872-83 (2015). ChIP ; Schizosaccharomyces pombe . Read more (PubMed: 25778913) »

See all 78 Publications for this product

Product Wall

Application Western blot
Sample Saccharomyces cerevisiae Cell lysate - whole cell (Whole cell lysate)
Gel Running Conditions Reduced Denaturing (15%)
Loading amount 60000 cells
Specification Whole cell lysate
Blocking step Odyssey blocking buffer as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 50% · Temperature: 20°C
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Submitted Aug 03 2016

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Na-citrate pH6
Sample Mouse Tissue sections (Liver, P5)
Specification Liver, P5
Permeabilization Yes - Triton 0,05%
Fixative Paraformaldehyde
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Submitted Oct 07 2014

Application Western blot
Loading amount 240000 cells
Gel Running Conditions Reduced Denaturing (10%)
Sample Mouse Cell lysate - whole cell (Primary Neural cells)
Specification Primary Neural cells
Treatment Supplemented with and without serum in the culture media.
Blocking step BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 28°C
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Submitted Sep 12 2014

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 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 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

Abcam guarantees this product to work in the species/application used in this Abreview.
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

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Application Western blot
Sample Mouse Cell lysate - whole cell (Mouse ES cells)
Loading amount 50 µg
Specification Mouse ES cells
Gel Running Conditions Reduced Denaturing (15% SDS-PAGE)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
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Submitted Nov 11 2011

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Application Western blot
Sample Human Cell lysate - whole cell (Hela Cells)
Loading amount 50 µg
Specification Hela Cells
Gel Running Conditions Reduced Denaturing (15% SDS-PAGE)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
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Submitted Nov 11 2011

Abcam has not validated the combination of species/application used in this Abreview.
Application Western blot
Sample Schistosoma mansoni Purified protein (1 adult worm)
Loading amount 1000 cells
Specification 1 adult worm
Gel Running Conditions Non-reduced Non-Denaturing (Native) (15% SDS page)
Blocking step Milk as blocking agent for 16 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C°C
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Submitted May 31 2011

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