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 applicationsICC, ICC/IF, PepArr, IHC-Fr, ChIP/Chip, WB, ChIP, IHC-P, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Rabbit, Cow, Human, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), 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).
    (Peptide available as ab32876)

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

Properties

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 Use at an assay dependent concentration.
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.
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).Can be blocked with Human Histone H3 (citrulline 2 + 8 + 17) peptide (ab32876). Abcam recommends using 3-5% milk as the blocking agent
ChIP Use at an assay dependent concentration.
IHC-P 1/100. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
Flow Cyt Use at an assay dependent concentration.

Target

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

  • Anti-Histone H3 (citrulline R2 + R8 + R17) antibody - ChIP Grade (ab5103) at 1/1000 dilution + Fruit fly (Drosophila melanogaster) tissue lysate - nuclear at 5 µg

    Secondary
    HRP-conjugated Goat anti-rabbit IgG polyclonal at 1/1000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15 kDa
    Observed band size : 15 kDa


    Exposure time : 1 minute

    This image is courtesy of an anonymous Abreview

    See Abreview

  • Anti-Histone H3 (citrulline R2 + R8 + R17) antibody - ChIP Grade (ab5103) at 1/1000 dilution + Caenorhabditis elegans embryo at 30 µg

    Secondary
    HRP-conjugated Goat anti-rabbit IgG polyclonal (undiluted)
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15 kDa
    Observed band size : 15 kDa
    Additional bands at : 10 kDa (possible non-specific binding).

    Exposure time : 1 minute

    This image is courtesy of an anonymous Abreview

    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

  • 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 Lonophore treated Whole Cell Lysate (ab80919) with with 5% BSA
    Lane 2 : HL60 (Human Caucasian promyelocytic leukaemia) DMSO and Calcium Lonophore treated Whole Cell Lysate (ab80919) with with 5% milk
    Lane 3 : HL60 (Human Caucasian promyelocytic leukaemia) DMSO and Calcium Lonophore treated Whole Cell Lysate (ab80919) 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 .

  • Chromatin immunoprecipitation using ab5103 on the pS2 promoter. Times are after stimulation by estrogen (Ul).
  • 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).
  • ab5103 staining Histone H3 (citrulline 2 + 8 + 17) in Mouse bone marrow cells by Immunocytochemistry/ Immunoflourescence. 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

  • 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.

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

This product has been referenced in:
  • Savchenko AS  et al. VWF-mediated leukocyte recruitment with chromatin decondensation by PAD4 increases myocardial ischemia/reperfusion injury in mice. Blood 123:141-8 (2014). ICC/IF ; Mouse . Read more (PubMed: 24200682) »
  • Deplus R  et al. Citrullination of DNMT3A by PADI4 regulates its stability and controls DNA methylation. Nucleic Acids Res N/A:N/A (2014). WB . Read more (PubMed: 24957603) »

See all 18 Publications for this product

Product Wall

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Caenorhabditis elegans Embryo (C. elegans embryo)
Specification C. elegans embryo
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Dec 18 2013

Application Immunocytochemistry
Blocking step Serum as blocking agent for 20 minute(s) · Concentration: 3% · Temperature: 23°C
Sample Human Cell (Human neutrophils (4 ´M calcium ionophore (A23187))
Specification Human neutrophils (4 ´M calcium ionophore (A23187)
Permeabilization Yes - 0.3% Triton X-100
Fixative Formaldehyde
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Submitted Dec 17 2013

Application Western blot
Loading amount 5 µg
Gel Running Conditions Reduced Denaturing (15%)
Sample Fruit fly (Drosophila melanogaster) Tissue lysate - nuclear (Fruit fly embryo)
Specification Fruit fly embryo
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Dec 17 2013

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 22°C
Sample Mouse Cell (Mouse bone marrow cells)
Specification Mouse bone marrow cells
Permeabilization Yes - 0.3% Triton-X 100
Fixative Paraformaldehyde
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Submitted Oct 11 2013

Thank you for contacting us. Here are somet ips regarding your ChIP experiment.


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...

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I have been able to confirm that you did in fact receive a vial of ab5103. It appears a technical issue during printing of the label led to the 'citH3' being left off the end of the target name.

Thank you for your email, and it's good to hear from you.

I've requested an update on the status of ab5103. Our UK office is closed for the day, but I expect to receive more information by tomorrow. I'll forward everything I find out as soon ...

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Thanks so much for your email.

I'm sorry to hear that the new lot is also not working well. We are looking into the issues with this antibody, but this can take some time to resolve.

I'm sending a free of charge replacement vial on ...

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Thank you very much for your reply.

I've received the single channel images from the lab, and these are attached. These show only the Ab5103 channel for the lots.

The staining looks more nuclear to me, but I'm interested to hear what...

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Thanks again for your patience while I've been in touch with the lab regarding these lots.

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 t...

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