Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)

Overview

  • Product name
    Anti-Histone H4 (di methyl K20) antibody - ChIP Grade
    See all Histone H4 primary antibodies
  • Description
    Rabbit polyclonal to Histone H4 (di methyl K20) - ChIP Grade
  • Tested applications
    Suitable for: IP, WB, ChIP, ICC/IF, IHC-Pmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human, Schizosaccharomyces pombe, Toxoplasma gondii
    Predicted to work with: Rat, a wide range of other speciesDoes not react with: Saccharomyces cerevisiae
  • Immunogen

    Synthetic peptide within Saccharomyces cerevisiae Histone H4 aa 1-100 (di methyl K20) conjugated to Keyhole Limpet Haemocyanin (KLH). The exact sequence is proprietary.
    (Peptide available as ab26213)

  • Positive control
    • WB: Calf thymus histone preparation and HeLa whole cell extract. IHC-P: FFPE human pancreas adenocarcinoma tissue.

Properties

Applications

Our Abpromise guarantee covers the use of ab9052 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
IP Use at an assay dependent concentration.
WB 1/2000. Detects a band of approximately 11 kDa.
ChIP Use at an assay dependent concentration. PubMed: 20829797
ICC/IF Use a concentration of 1 µg/ml.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

Target

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

  • ICC/IF image of ab9052 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab9052 at 1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • Histone H4 (di methyl K20) was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to Histone H4 (di methyl K20) and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
    The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
    Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab9052.
    Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
    Band: 14kDa: Histone H4 (di methyl K20).

  • All lanes : Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052) at 1/2000 dilution

    Lane 1 : yH4 17-24 peptide
    Lane 2 : Unmodified yH4 17-24 peptide
    Lane 3 : Mono methyl K20 yH4 17-24 peptide
    Lane 4 : Di methyl K20 yH4 17-24 peptide
    Lane 5 : Tri methyl K20 yH4 17-24 peptide

    Blocking peptides at 1 µg/ml per lane.


    Performed under reducing conditions.

    This image is courtesy of Steve Sanders, Tony Kouzarides lab, University of Cambridge

  • All lanes : Upper blot - Histone H4 antibody (gift of A. Verreault) Lower blot - Rabbit polyclonal to Histone H4 di methyl K20 (ab9052) at 1/2000

    Lane 1 : S.cerevisiae extract
    Lane 2 : S.pombe extract
    Lane 3 : S.pombe rH4
    Lane 4 : Drosophila rH4
    Lane 5 : Histones (Roche)

    Di methylation at K20 is seen in S. pombe and in calf thymus (Roche). S. cerevisiae lacks K20 (di) methylation.

  • IHC image of ab9052 staining in human pancreas adenocarcinoma 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 ab9052, 1µg/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.

References for Anti-Histone H4 (di methyl K20) antibody - ChIP Grade (ab9052)

This product has been referenced in:
  • Gursoy-Yuzugullu O  et al. Epigenetic therapy with inhibitors of histone methylation suppresses DNA damage signaling and increases glioma cell radiosensitivity. Oncotarget 8:24518-24532 (2017). WB ; Human . Read more (PubMed: 28445939) »
  • Kidder BL  et al. SMYD5 regulates H4K20me3-marked heterochromatin to safeguard ES cell self-renewal and prevent spurious differentiation. Epigenetics Chromatin 10:8 (2017). CHIPseq ; Mouse . Read more (PubMed: 28250819) »

See all 19 Publications for this product

Product Wall

Application
Immunocytochemistry/ Immunofluorescence
Sample
Caenorhabditis elegans Cell (Gut cell & embryos)
Specification
Gut cell & embryos
Fixative
Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 10 2015

Application
Western blot
Sample
Human Cell lysate - other (HeLa, mouse ES)
Gel Running Conditions
Reduced Denaturing (4-12 % SDS PAGE)
Loading amount
2 µg
Specification
HeLa, mouse ES
Blocking step
Milk as blocking agent for 18 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 4°C
Username

Abcam user community

Verified customer

Submitted Aug 04 2015

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
Username

Abcam user community

Verified customer

Submitted Oct 07 2014

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Human Cell lysate - whole cell (multiple myeloma cells)
Loading amount
50 µg
Specification
multiple myeloma cells
Gel Running Conditions
Reduced Denaturing (10-20% tris glycine)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Nov 21 2012

Abcam has not validated the combination of species/application used in this Abreview.
Application
Western blot
Sample
Caenorhabditis elegans Tissue lysate - nuclear (C. elegans)
Loading amount
10 µg
Specification
C. elegans
Treatment
0, 75Gy r-ray
Gel Running Conditions
Reduced Denaturing (12%)
Blocking step
Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Ms. Jinsun Ryu

Verified customer

Submitted Sep 12 2012

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

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