Anti-Histone H3 (citrulline R2 + R8 + R17) antibody - ChIP Grade (ab5103)

Overview

  • Product nameAnti-Histone H3 (citrulline R2 + R8 + R17) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (citrulline R2 + R8 + R17) - ChIP Grade
  • Specificityab5103 detects a 17 kDa band in single lane Western Blot. Peptide inhibition in Western Blot hasn't been processed. Modification specificity is determined by Peptide Array. ab5103 binds strongly to Histone H3 citrulline 2 + 8 + 17 peptide.
  • Tested applicationsSuitable for: ICC/IF, PepArr, IHC-Fr, Flow Cyt, ChIP/Chip, WB, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Cow, Human, Monkey
    Predicted to work with: a wide range of other species
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 1-100 (citrulline R2 + R8 + R17) conjugated to Keyhole Limpet Haemocyanin (KLH). Also SwissProt: P84243, Q71DI3, Q16695, Q6NXT2.
    Database link: P68431
    (Peptide available as ab32876)

  • Positive control
    • This antibody gave a positive signal in HL60 Whole Cell Lysate - DMSO and Calcium Ionophore treated. In WB ab5103 only recognizes human or bovine histone H3 when PADI4 and calcium are added.

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 bufferpH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Batches of this product that have a concentration < 1mg/ml may have BSA added as a stabilising agent. If you would like information about the formulation of a specific lot, please contact our scientific support team who will be happy to help.
  • Concentration information loading...
  • PurityImmunogen affinity purified
  • ClonalityPolyclonal
  • IsotypeIgG
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab5103 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
ICC/IF Use at an assay dependent concentration. PubMed: 20733033
PepArr Use a concentration of 0.2 - 0.02 µg/ml.
IHC-Fr Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

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

ChIP/Chip Use at an assay dependent concentration.
WB Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa). Abcam recommends using 3-5% milk as the blocking agent
ChIP Use at an assay dependent concentration.

Target

  • RelevanceCore 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- modifications of histones, also called histone code, and nucleosome remodeling. P68431 and Q71DI3 are expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation. Q16695 is expressed in tesicular cells. Q6NXT2 is specifically expressed in the seminiferous tubules of testis and this Hominid-specific H3.5/H3F3C preferentially colocalizes with euchromatin, and it is associated with actively transcribed genes. P84243 is a variant histone H3 which replaces conventional H3 in a wide range of nucleosomes in active genes. Constitutes the predominant form of histone H3 in non-dividing cells and is incorporated into chromatin independently of DNA synthesis. Deposited at sites of nucleosomal displacement throughout transcribed genes, suggesting that it represents an epigenetic imprint of transcriptionally active chromatin. Post-translational modification. Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
  • Cellular localizationNuclear
  • Database links
  • Alternative names
    • Cit Histone H3 antibody
    • Citrullated antibody
    • citrullin antibody
    • Citrullinated Histone H3 antibody
    • citrulline antibody
    • H3 3 like sequence MH921 antibody
    • H3 3A antibody
    • H3 histone antibody
    • H3 histone family member E pseudogene antibody
    • H3F3 antibody
    • HIST3H3 antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3 Citrullated antibody
    • Histone H3.1 antibody
    • Histone H3.1t antibody
    • Histone H3.2 antibody
    • Histone H3.3 antibody
    • Histone H3.3C antibody
    • Histone H3.5 antibody
    see all

Anti-Histone H3 (citrulline R2 + R8 + R17) antibody - ChIP Grade images

  • All batches of ab5103 are tested in Peptide Array against peptides to different Histone H3 modifications. Six dilutions of each peptide are printed on to the Peptide Array in triplicate and results are averaged before being plotted on to a graph. Results show strong binding to Histone H3 - citrulline 2 + 8 + 17 peptide (ab32876), indicating that this antibody specifically recognises the Histone H3 - citrulline 2 + 8 + 17 modifications.

    ab32876 - Histone H3 - citrulline 2 + 8 + 17

    ab17566 - Histone H3 - unmodified

  • All lanes : Anti-Histone H3 (citrulline R2 + R8 + R17) antibody - ChIP Grade (ab5103) at 0.2 µg/ml

    Lane 1 : HL60 whole cell lysate (negative control)
    Lane 2 : HL60 whole cell lysate + DMSO (solvent control)
    Lane 3 : HL60 whole cell lysate + DMSO + Calcium Ionophore (positive control)

    Lysates/proteins at 20 µg per lane.

    Secondary
    Goat anti Rabbit IR680 at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size : 15 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)

    Loading Control: GAPDH

    This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using Licor blocking buffer before being incubated with ab5103 overnight at 4°C. Antibody binding was detected using Goat anti Rabbit IR680 secondary at a 1:10,000 dilution for 1hr at room temperature and then imaged using the Licor Odyssey CLx.

     

  • Chromatin immunoprecipitation using ab5103 on the pS2 promoter. Times are after stimulation by estrogen (Ul).
  • ab5103 staining Histone H3 (citrulline 2 + 8 + 17) in Mouse bone marrow cells by Immunocytochemistry/ Immunofluorescence. Cells were fixed in formaldehyde and permeabilized in 0.1% Triton X-100 prior to blocking in 5% Goat serum for 2 hours at 25°C. The primary antibody was diluted 1/250 in PBS and incubated with the sample for 12 hours at 4°C. The secondary antibody was Alexa Fluor® 488-conjugated Goat anti-Rabbit polyclonal, diluted 1/500.
    Nuclei were counterstained blue with DAPI.

    See Abreview



  • Predicted band size : 15 kDa

    Cuthbert, G.L. et al, (2004) Cell 118, 545-553

    Rabbit polyclonal to Histone H3 (citrulline 2 + 8 + 17) used at 1/2000 dilution, after blocking with TBST 5% BSA. Purified histones run out with approximately 250 ng of each histone.

    Lanes 1-3 contain Histone H3 (250 ng per lane)
    Lane 1: PADI4 + Calcium
    Lane 2: H3 + PADI4
    Lane 3: H3 + PADI4 + Calcium

    Lanes 4-5 contain bulk histones (250 ng per lane)
    Lane 4: PADI4
    Lane 5: PADI4 + Calcium

    Lane 6: MCF7 cell extract
    Lane 7: MCF7 cell extract (HA-PADI4)

    Secondary antibody : anti-rabbit HRP from Sigma.
    In WB ab5103 only recognizes human or bovine histone H3 when PADI4 and calcium are added.

  • ICC/IF image of ab5103 stained human HeLa cells. The cells were PFA fixed (10 min), permabilised in TBS-T (20 min) and incubated with the antibody (ab5103, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to quench autofluorescence and block non-specific protein-protein interactions. The secondary antibody (green) was Alexa Fluor® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red). DAPI was used to stain the cell nuclei (blue).

  • All lanes : Anti-Histone H3 (citrulline R2 + R8 + R17) antibody - ChIP Grade (ab5103) at 1 µg/ml

    Lane 1 : HL60 (Human Caucasian promyelocytic leukaemia) DMSO and Calcium Ionophore treated Whole Cell Lysate with with 5% BSA
    Lane 2 : HL60 (Human Caucasian promyelocytic leukaemia) DMSO and Calcium Ionophore treated Whole Cell Lysate with with 5% milk
    Lane 3 : HL60 (Human Caucasian promyelocytic leukaemia) DMSO and Calcium Ionophore treated Whole Cell Lysate with with 3% milk

    Lysates/proteins at 10 µg per lane.

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/10000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)


    Exposure time : 30 seconds

    Abcam recommends using milk as the blocking agent. Abcam welcomes customer feedback and would appreciate any comments regarding this product and the data presented above .

     

    Secondary antibody - goat anti-rabbit HRP (ab97051)

References for Anti-Histone H3 (citrulline R2 + R8 + R17) antibody - ChIP Grade (ab5103)

This product has been referenced in:
  • Zenaro E  et al. Neutrophils promote Alzheimer's disease-like pathology and cognitive decline via LFA-1 integrin. Nat Med N/A:N/A (2015). Read more (PubMed: 26214837) »
  • Mizurini DM  et al. Salivary Thromboxane A2-Binding Proteins from Triatomine Vectors of Chagas Disease Inhibit Platelet-Mediated Neutrophil Extracellular Traps (NETs) Formation and Arterial Thrombosis. PLoS Negl Trop Dis 9:e0003869 (2015). Mouse . Read more (PubMed: 26110417) »

See all 26 Publications for this product

Product Wall

Application Western blot
Sample Human Cell lysate - whole cell (MCF-7, Hep G2)
Gel Running Conditions Reduced Denaturing
Loading amount 25 µg
Specification MCF-7, Hep G2
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
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Abcam user community

Verified customer

Submitted Aug 19 2016

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Kidney)
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: 10 mM Citrate
Permeabilization No
Specification Kidney
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Fixative Paraformaldehyde
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Abcam user community

Verified customer

Submitted Oct 06 2015

Application Western blot
Sample Mouse Tissue lysate - whole (Kidney)
Gel Running Conditions Reduced Denaturing (4-12%)
Loading amount 20 µg
Specification Kidney
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Dr. Wesley Konsavage

Verified customer

Submitted Oct 05 2015

Application Western blot
Loading amount 15 µg
Gel Running Conditions Reduced Denaturing
Sample Mouse Cell lysate - whole cell (hepatocytes)
Specification hepatocytes
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

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Verified customer

Submitted Dec 26 2014

The image shown using PADI4 and Calcium on our datasheet is copied from 2004 Cell paper by Kouzarides’ lab.

This experiment was not actually performed in the abcam labs and therefore I’m afraid that I can’t add much clarific...

Read More

HeLa Cells:
For 3.5 x 10e6 HeLa cells per plate, we pool 2 plates per falcon tube (in about 16ml PBS). We then centrifuge the cells @ 4°C for 5mins at 3000rpm. The PBS is then aspirated off and the cells re-suspended into 2.8ml of ChIP lysis buffe...

Read More

I've attached the ICC images that we have for the lots that you requested. The staining in both images is more nuclear than the previous image I sent to you.

The staining was performed using the protocol that is shown underneath the image on ...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Skin)
Specification Skin
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: EDTA pH 9.0
Permeabilization No
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Verified customer

Submitted Jun 01 2012

The link to our indirect ELISA protocol is:
http://www.abcam.com/index.html?pageconfig=resource&rid=11389

Due to the various other modifications to this protein, it is likely that the shifted bands represent modified Histone H3 that are present in this sample, but not your other samples. Such bands are also observed in our own validation WB and can be bloc...

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