Anti-Histone H3 (mono methyl R2) antibody - ChIP Grade (ab15584)

Overview

  • Product nameAnti-Histone H3 (mono methyl R2) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (mono methyl R2) - ChIP Grade
  • Tested applicationsIP, ChIP/Chip, Dot Blot, IHC-P, PepArr, Flow Cyt, WB, ChIPmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human, Saccharomyces cerevisiae
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 to the C-terminus Histone H3, mono methylated at R2.

  • Positive control
    • Calf thymus histone, C3H10T1/2 and 293 lysates. In ICC/IF this antibody gave a positive result in HeLa cell line.

Properties

Applications

Our Abpromise guarantee covers the use of ab15584 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 a concentration of 5 µg/ml.
ChIP/Chip Use at an assay dependent concentration.
Dot Blot Use at an assay dependent concentration.
IHC-P 1/80. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
PepArr Use a concentration of 0.2 - 2 µg/ml.
Flow Cyt Use at an assay dependent concentration.
WB Use a concentration of 0.5 - 2 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 17 kDa).Can be blocked with Histone H3 (mono methyl R2) peptide (ab1775).
ChIP Use 2-4 µg for 25 µg of chromatin.

Target

  • FunctionCore 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 similaritiesBelongs to the histone H3 family.
  • Developmental stageExpressed 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 localizationNucleus. Chromosome.
  • Information by UniProt
  • Database links
  • Alternative names
    • H3 histone family, member J antibody
    • FLJ92264 antibody
    • H3 histone antibody
    • H3 histone antibody
    • H3 histone family, member A antibody
    • H3 histone family, member B antibody
    • H3 histone family, member C antibody
    • H3 histone family, member D antibody
    • H3 histone family, member F antibody
    • H3 histone family, member H antibody
    • H3 histone family, member I antibody
    • H3 histone family, member K antibody
    • H3 histone family, member L antibody
    • H3 histone, family 3A antibody
    • H3.3A antibody
    • H3/a antibody
    • H3/b antibody
    • H3/c antibody
    • H3/d antibody
    • h3/f antibody
    • H3/h antibody
    • H3/i antibody
    • H3/j antibody
    • H3/k antibody
    • H3/l antibody
    • H31_HUMAN antibody
    • H3F1K antibody
    • H3F3 antibody
    • H3F3 antibody
    • H3FA antibody
    • H3FB antibody
    • H3FC antibody
    • H3FD antibody
    • H3FF antibody
    • H3FH antibody
    • H3FI antibody
    • H3FJ antibody
    • H3FK antibody
    • H3FL antibody
    • HIST1H3A antibody
    • HIST1H3B antibody
    • HIST1H3C antibody
    • HIST1H3D antibody
    • HIST1H3E antibody
    • HIST1H3F antibody
    • HIST1H3G antibody
    • HIST1H3H antibody
    • HIST1H3I antibody
    • HIST1H3J antibody
    • HIST3H3 antibody
    • HIST3H3 antibody
    • Histone 1, H3a antibody
    • Histone 1, H3b antibody
    • Histone 1, H3c antibody
    • Histone 1, H3d antibody
    • Histone 1, H3e antibody
    • Histone 1, H3f antibody
    • Histone 1, H3g antibody
    • Histone 1, H3h antibody
    • Histone 1, H3i antibody
    • Histone 1, H3j antibody
    • Histone cluster 1, H3a antibody
    • Histone cluster 1, H3b antibody
    • Histone cluster 1, H3c antibody
    • Histone cluster 1, H3d antibody
    • Histone cluster 1, H3e antibody
    • Histone cluster 1, H3f antibody
    • Histone cluster 1, H3g antibody
    • Histone cluster 1, H3i antibody
    • Histone cluster 1, H3j antibody
    • Histone H 3 antibody
    • Histone H3.1 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
    • R2 antibody
    see all

Anti-Histone H3 (mono methyl R2) antibody - ChIP Grade images

  • Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of  ab15584 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach). Primers and probes are located in the first kb of the transcribed region.     
  • All batches of ab15584 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 - mono methyl R2 peptide (ab1775), indicating that this antibody specifically recognises the Histone H3 - mono methyl R2 modification.

    1.   ab1775 – Histone H3 - mono methyl R2 peptide
    2.   ab38488 – Histone H3 - symmetric di methyl R2
    3.   ab2853 – Histone H3  - asymmetric di methyl R2
    4.   ab17566 – Histone H3  - non-modified
    5.   ab33971 – Histone H3  - mono methyl R8
    6.   ab13845 – Histone H3  - mono methyl R17
    7.   ab16935 – Histone H3  - asymmetric di methyl R17
    8.   ab32948 – Histone H3  - symm

  • Anti-Histone H3 (mono methyl R2) antibody - ChIP Grade (ab15584) at 1 µg/ml + Calf Thymus Histone Preparation Nuclear Lysate at 0.5 µg

    Secondary
    Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

    Performed under reducing conditions.

    Predicted band size : 17 kDa


    Exposure time : 3 minutes
  • All lanes : Anti-Histone H3 (mono methyl R2) antibody - ChIP Grade (ab15584) at 0.5 µg/ml

    Lane 1 : Calf thymus histone at 0.4 µg
    Lane 2 : C3H10T1/2 histones at 0.4 µg
    Lane 3 : 293 histones at 0.4 µg
    Lane 4 : Yeast extracts from 1 x 106 cells


    Predicted band size : 17 kDa
    ab15584 specifically recognises mono-methylated Histone H3 at R2 in calf thymus (lane1), mouse (lane2) and human (lane3) histone preparations as well as in yeast whole cell extract (lane4).
  • Histone H3 was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H3 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 ab15584.
    Secondary: Clean-Blot IP Detection Reagent (HRP) at 1/500 dilution.
    Band: 17kDa; Histone H3
  • Image courtesy of Human Protein Atlas

    ab15584 staining histone H3 mono methyl R2 in human testis, showing a distinct and strong nuclear staining pattern in ductus seminiferus and leydig cells. Paraffin embedded human skin tissue was incubated with ab15584 (1/80 dilution) for 30 mins at room temperature. Antigen retrieval was performed by heat induction in citrate buffer pH 6.

    ab15584 was tested in a tissue microarray (TMA) containing a wide range of normal and cancer tissues as well as a cell microarray consisting of a range of commonly used, well characterised human cell lines. Further results for this antibody can be found at www.proteinatlas.org

    .
  • ICC/IF image of ab15584 stained HeLa cells. The cells were 100% methanol fixed (5 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab15584, 5µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, a goat anti-rabbit DyLight® 488 (IgG; H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM.

References for Anti-Histone H3 (mono methyl R2) antibody - ChIP Grade (ab15584)

This product has been referenced in:
  • Majocchi S  et al. Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes. Nucleic Acids Res N/A:N/A (2013). ChIP . Read more (PubMed: 24071586) »
  • Chignola F  et al. The solution structure of the first PHD finger of autoimmune regulator in complex with non-modified histone H3 tail reveals the antagonistic role of H3R2 methylation. Nucleic Acids Res 37:2951-61 (2009). WB ; Human . Read more (PubMed: 19293276) »

See all 5 Publications for this product

Product Wall

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - eBioscience Permeabilization Buffer
Sample Human Cell (Differentiated Haematopoietic Stem Cells)
Specification Differentiated Haematopoietic Stem Cells
Gating Strategy Isotype negative control (white)
Preparation Cell harvesting/tissue preparation method: Typsin-EDTA Cell Dissociation
Sample buffer: PBS and 10% FBS
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Submitted Feb 19 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (12)
Sample Human Cell lysate - whole cell (Human HeLa cells)
Specification Human HeLa cells
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
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Submitted Nov 11 2013

Thank you for contacting Abcam regarding ab15584. Please contact Abcam for further immunogen information. We are happy to provide it per our Proprietary Immunogen Policy. I am sorry to hear that you have some concerns regarding the specificity a...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Saccharomyces cerevisiae Cell lysate - whole cell (BY4741 strain)
Loading amount 100000 cells
Specification BY4741 strain
Blocking step BSA as blocking agent for 2 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Oct 23 2007

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