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

Overview

  • Product name
    Anti-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
  • Host species
    Rabbit
  • Specificity
    ab5103 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 applications
    Suitable 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.
  • General notes

    Learn about ChIP assay kits, other ChIP antibodies, protocols and more in the ChIP assay guide.

    Abcam recommended secondaries - Goat Anti-Rabbit HRP (ab205718) and Goat Anti-Rabbit Alexa Fluor® 488 (ab150077). See other anti-rabbit secondary antibodies that can be used with this antibody.

Properties

  • Form
    Liquid
  • Storage instructions
    Shipped 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 buffer
    pH: 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...
  • Purity
    Immunogen affinity purified
  • Clonality
    Polyclonal
  • Isotype
    IgG
  • 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

We recommend Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody.

ChIP Use at an assay dependent concentration.

Target

  • Function
    Core 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 similarities
    Belongs to the histone H3 family.
  • Developmental stage
    Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
  • Post-translational
    modifications
    Acetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
    Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
    Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters.
    Methylation at Lys-5 (H3K4me), Lys-37 (H3K36me) and Lys-80 (H3K79me) are linked to gene activation. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are linked to gene repression. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin.
    Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin.
    Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (By similarity). 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.
  • Cellular localization
    Nucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • H3 histone family member E pseudogene antibody
    • H3 histone family, member A antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3F3 antibody
    • H3FA antibody
    • Hist1h3a antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • HIST3H3 antibody
    • histone 1, H3a antibody
    • Histone cluster 1, H3a antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3.1 antibody
    • Histone H3/a antibody
    • Histone H3/b antibody
    • Histone H3/c antibody
    • Histone H3/d antibody
    • Histone H3/f antibody
    • Histone H3/h antibody
    • Histone H3/i antibody
    • Histone H3/j antibody
    • Histone H3/k antibody
    • Histone H3/l antibody
    see all

Images

  • Chromatin immunoprecipitation using ab5103 on the pS2 promoter. Times are after stimulation by estrogen (Ul).
  • 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
    All lanes : 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.

     

  • 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

  • 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

  • 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
    All lanes : 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 .

    Blots were developled with Goat Anti-Rabbit IgG H&L (HRP) (ab97051) secondary antibody

References

This product has been referenced in:
  • Nomura K  et al. Citrullinated Histone H3: Early Biomarker of Neutrophil Extracellular Traps in Septic Liver Damage. J Surg Res 234:132-138 (2019). Read more (PubMed: 30527465) »
  • Murthy P  et al. Enhanced Neutrophil Extracellular Trap Formation in Acute Pancreatitis Contributes to Disease Severity and Is Reduced by Chloroquine. Front Immunol 10:28 (2019). Read more (PubMed: 30733719) »
See all 140 Publications for this product

Customer reviews and Q&As

1-10 of 25 Abreviews or Q&A

Application
Western blot
Sample
Human Cell lysate - nuclear (Vascular smooth muscle cell)
Gel Running Conditions
Reduced Denaturing
Loading amount
20 µg
Specification
Vascular smooth muscle cell
Blocking step
Milk as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 21°C

Abcam user community

Verified customer

Submitted Dec 18 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Human Cell (Vascular smooth muscle cell)
Permeabilization
Yes - NP40
Specification
Vascular smooth muscle cell
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 21°C
Fixative
Formaldehyde

Abcam user community

Verified customer

Submitted Dec 18 2018

Abcam guarantees this product to work in the species/application used in this Abreview.
Application
Western blot
Sample
Mouse Cell lysate - whole cell (RAW 264.7 cells)
Gel Running Conditions
Reduced Denaturing (4-20%)
Loading amount
25 µg
Specification
RAW 264.7 cells
Blocking step
Milk as blocking agent for 1 hour(s) and 30 minute(s) · Concentration: 5% · Temperature: RT°C

Abcam user community

Verified customer

Submitted Jun 07 2018

Application
Immunocytochemistry/ Immunofluorescence
Sample
Mouse Cell (macrophage cell line)
Permeabilization
Yes - 0.1% Triton in PBS for 5 min
Specification
macrophage cell line
Blocking step
Serum as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: RT°C
Fixative
Paraformaldehyde

Abcam user community

Verified customer

Submitted Jun 04 2018

Application
Immunohistochemistry (Frozen sections)
Sample
Mouse Cell (Lung)
Permeabilization
Yes - Triton 0.1%
Specification
Lung
Blocking step
BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 37°C
Fixative
Zinc fixative

Abcam user community

Verified customer

Submitted Jul 26 2017

Application
ELISA
Sample
Human Serum (EDTA Plasma Samples)
Specification
EDTA Plasma Samples
Blocking step
BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: 21°C
Type
Direct

Herr Dr. Thomas Scherz

Verified customer

Submitted Apr 28 2017

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

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

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

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

Abcam user community

Verified customer

Submitted Dec 26 2014

1-10 of 25 Abreviews or Q&A

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

Sign up