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.

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 A antibody
    • H3/A antibody
    • H31_HUMAN antibody
    • H3FA antibody
    • Hist1h3a antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • histone 1, H3a antibody
    • Histone cluster 1, H3a 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

  • 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

  • Chromatin immunoprecipitation using ab5103 on the pS2 promoter. Times are after stimulation by estrogen (Ul).
  • 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:
  • Fetz AE  et al. Localized Delivery of Cl-Amidine From Electrospun Polydioxanone Templates to Regulate Acute Neutrophil NETosis: A Preliminary Evaluation of the PAD4 Inhibitor for Tissue Engineering. Front Pharmacol 9:289 (2018). Read more (PubMed: 29643810) »
  • Pircher J  et al. Cathelicidins prime platelets to mediate arterial thrombosis and tissue inflammation. Nat Commun 9:1523 (2018). Read more (PubMed: 29670076) »
See all 104 Publications for this product

Customer reviews and Q&As

1-10 of 23 Abreviews or Q&A

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

Answer

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 clarification to what’s already described in the original paper:

http://www.ncbi.nlm.nih.gov/pubmed/15339660?dopt=Abstract



It looks that left-hand side and middle panels are in-vitro modification of H3 using purified recomb PADI4 that deposits the cit-modification and active only in the presence of Ca++

Left-hand side panel is overexpression of PADI4 in MCF7 cells.



“The Cit-H3 antibody http://www.sciencedirect.com/science/article/pii/S0092867404007998#FIG3 was raised against a peptide corresponding to the tail of H3 with citrulline present in the place of R2, R8, and R17 http://www.sciencedirect.com/science/article/pii/S0092867404007998#FIG3. http://www.sciencedirect.com/science/article/pii/S0092867404007998#FIG3 shows that the Cit-H3 antibody recognizes the faster migrating deiminated H3, which is generated when PADI4 is incubated with H3. No reactivity is observed with unmodified H3 http://www.sciencedirect.com/science/article/pii/S0092867404007998#FIG3 or with other histones treated by PADI4 http://www.sciencedirect.com/science/article/pii/S0092867404007998#FIG3. Use of this antibody in Western blots indicates that H3 has virtually undetectable levels of citrulline in cultured MCF-7 cells (http://www.sciencedirect.com/science/article/pii/S0092867404007998#FIG3, lane 1). However, when PADI4 is overexpressed in these cells, detection of deiminated H3 by the Cit-H3 antibody increases substantially (http://www.sciencedirect.com/science/article/pii/S0092867404007998#FIG3, lane 2). These results confirm that endogenous H3 tail arginine residues are deiminated by the action of PADI4.”



Experimental Procedures

In Vitro Reactions

GST.PADI4 constructs were expressed at 30°C in 2TY media in 0.1 mM IPTG from pGEX6p and purified using glutathione-Sepharose resin. Deimination reactions were carried out using bead-bound enzyme in 50 mM HEPES [pH 7.5], 2 mM DTT, and 10 mM CaCl2 at 30°C. Substrates were bulk calf thymus histones (Sigma), purified calf thymus histone H3 (Roche), recombinant histone H3 and tailless histone H3 lacking residues 1–26, and various peptides. Peptides described in the manuscript were synthesized by Graham Bloomberg at the University of Bristol.

GST.CARM1 was expressed and purified as above. GST.CARM1 was eluted using 50 mM reduced glutathione then dialyzed against 10% glycerol in PBS. Methylation reactions were carried out in 5% glycerol, PBS with [3H]S-adenosyl-L-methionine. CARM1 peptide reactions were bound to p81 cation exchange membrane (Whatman), washed with 50 mM carbonate buffer [pH 9.2], and incorporated radioactivity measured by scintillation counting http://www.sciencedirect.com/science/article/pii/S0092867404007998#BIB3.

HEK293 and MCF-7 cells were maintained in Dulbecco's modified Eagle's medium (DMEM, Gibco-BRL) supplemented with 10% fetal calf serum (FCS, Gibco-BRL) at 37°C and 5% CO2. Cells were transfected with pcDNA3.HA.PADI4 using FuGENE transfection reagent (Roche) according to the manufacturer's instructions. 48 hr posttransfection cells were lysed in 50 mM HEPES [pH 7.5], 2 mM DTT, 150 mM NaCl, 0.5% NP-40, and HA.PADI4 was immunoprecipitated using an anti-HA antibody (Abcam ab9110). Immunoprecipitated bead-bound protein was washed with lysis buffer and reactions carried in vitro out as above. Whole-cell extracts were made by lysing MCF-7 cells in RIPA buffer (50 mM Tris [pH 8.0], 150 mM NaCl, 1% NP-40, 0.5% deoxycholate).

Antibodies

Polyclonal α-PADI4 was raised by immunizing a rabbit with purified GST-PADI4 then affinity purifying the antibody from whole serum with GST-PADI4 http://www.sciencedirect.com/science/article/pii/S0092867404007998#BIB11. Polyclonal α-modified citrulline was purchased from Upstate (Cat.no.17-347) and the modification of membrane bound citrulline carried out according to the supplier's instructions. The monoclonal antibody recognizing context-independent monomethyl arginine was purchased from Abcam (ab415), as was the antibody against dimethyl R17 of H3 (ab8284). The polyclonal citH3 antibody was raised in collaboration with Abcam (ab5103) by immunizing a rabbit with a peptide of the sequence ACitTKQTACitKSTGGKAPCitKQLA and purifying the antibody against the same peptide. The monoclonal antibody against RNA polymerase II was purchased from Covance (Cat. no. MMS-126R-500).

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Answer

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 buffer and incubated on ice for 10mins. Sonicate after these 10mins.

Sonication:
We never use ethanol, just ice cold water. It is important though that there is no ice in the water. We use a Bioruptor with probes. The cells are sonicated for 40mins at intervals of 5mins, changing the water in between each 5min sonication. The 5min sonication is 30secs sonication and then 30sec rest (5 times).

There is no real answer to avoiding foaming during sonication as the lysis buffer can cause this. What I would recommend though, is to check for foaming/bubbles in between the 5min sonications and if there are bubbles etc, transfer the supernatant to a new tube, leaving the bubbles/foam behind - this tends to work.

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1-10 of 23 Abreviews or Q&A

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