Anti-Histone H3 (asymmetric di methyl R17) antibody - ChIP Grade (ab8284)

Overview

  • Product nameAnti-Histone H3 (asymmetric di methyl R17) antibody - ChIP GradeSee all Histone H3 primary antibodies ...
  • Description
    Rabbit polyclonal to Histone H3 (asymmetric di methyl R17) - ChIP Grade
  • SpecificityPeptide competition experiments confirmed that the antibody recognises specifically methylated R17 in H3 and not unmethylated H3 or methylated R3 in H4 (see figure 1). In whole cell extract the antibody recognises specifically only the methylated histone H3 protein band (see figure 2) Further, the antibody doesn't crossreact with the C-terminal methylation sites of CARM1 in histone H3. In IHC on paraffin-embedded sections of human tonsil, the antibody shows nuclear staining across most nuclei. Slight batch to batch variation is observed, but no more than 50% cross reactivity with symmetric di methyl R17 peptide is allowed.
  • Tested applicationsChIP/Chip, PepArr, IHC-P, ICC/IF, ChIP, Dot Blot, IP, WBmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cow, Human, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Toxoplasma gondii
    Predicted to work with: all Mammals
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 1-100 (asymmetric di methyl R17) conjugated to Keyhole Limpet Haemocyanin (KLH).
    (Peptide available as ab16935)

  • Positive control
    • This antibody gave a positive signal in HeLa Histone Preparation Nuclear whole cell lysate. It also gave a positive result in MCF7 cell line.
  • General notes

    The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on purified histones. This antibody recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Bauer et al (2001) have shown by using this antibody that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is stimulated by estradiol and TPA. Coincident with the appearance of methylated R17, the CARM1 methyltransferase is found associated with the histones on the pS2 gene. Together these results demonstrate that the CARM1 methyltransferase is recruited to an active promoter and that CARM1-mediated methylation of histone H3 at R17 takes place in vivo during this active state.

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
    Note: 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
  • Primary antibody notes The nuclear hormone receptor co-activator CARM1 has the potential to methylate histone H3 at arginine residues in vitro. The methyltransferase activity of CARM1 is necessary for its co-activator functions in transient transfection assays. However, the role of this methyltransferase in vivo is unclear, given that methylation of arginines is not easily detectable on purified histones. This antibody recognizes methylated arginine 17 (R17) of histone H3, the major site of methylation by CARM1. Bauer et al (2001) have shown by using this antibody that methylated R17 exists in vivo. Chromatin immunoprecipitation analysis shows that R17 methylation on histone H3 is dramatically upregulated when the estrogen receptor-regulated pS2 gene is stimulated by estradiol and TPA. Coincident with the appearance of methylated R17, the CARM1 methyltransferase is found associated with the histones on the pS2 gene. Together these results demonstrate that the CARM1 methyltransferase is recruited to an active promoter and that CARM1-mediated methylation of histone H3 at R17 takes place in vivo during this active state.
  • Clonality Polyclonal
  • IsotypeIgG
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab8284 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
ChIP/Chip Use at an assay dependent dilution.
PepArr Use a concentration of 0.002 - 0.0002 µg/ml.
IHC-P Use at an assay dependent dilution.
ICC/IF Use a concentration of 0.1 µg/ml.
ChIP Use at an assay dependent dilution.
Dot Blot Use at an assay dependent dilution.
IP Use at an assay dependent dilution.
WB 1/1000 - 1/2000. Can be blocked with Histone H3 (asymmetric di methyl R17) peptide (ab16935).

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
    see all

Anti-Histone H3 (asymmetric di methyl R17) antibody - ChIP Grade images

  • All batches of ab8284 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 - asymmetric di methyl R17 peptide (ab16935), indicating that this antibody specifically recognises the Histone H3 - asymmetric di methyl R17 modification.

    1. ab16935 - Histone H3 - asymmetric di methyl R17
    2. ab32948 - Histone H3 - symmetric di methyl R17
    3. ab14663 - Histone H3 - unmodified
  • Anti-Histone H3 (asymmetric di methyl R17) antibody - ChIP Grade (ab8284) at 1 µg/ml + HeLa Histone Preparation Nuclear Lysate at 2.5 µg/ml

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution
    developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 15.4 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)
    Additional bands at : 55 kDa,60 kDa,90 kDa. We are unsure as to the identity of these extra bands.

    Exposure time : 2 minutes
  • Total U2OS cell extract was western blotted using the anti-Me-R17H3 antibody. The asterisk indicates methylated histone H3. The left panel shows presence of core histones (indicated on the left) by Coomassie Blue staining. Molecular weights are indicated on the right.

  • A dot blot was performed using unmodified peptide (lane 1), Histone H3 mono methyl R17 peptide (lane 2), Histone H3 asymmetric di methyl R17 peptide (lane 3) and Histone H3 symmetric di methyl R17 peptide (lane 4). The dot blot indicates that ab8284 is specific to Histone H3 asymmetric di methyl R17.
  • ab8284 was used in immunohistochemistry with paraffin embedded sections of human tonsil, using DAB as a chromogen (brown). Counterstaining of nuclei was performed with haemotoxylin (blue).

    Staining is seen confined to the nucleus.

  • ICC/IF image of ab8284 stained MCF7 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 (ab8284, 0.1µg/ml) overnight at +4°C. The secondary antibody (green) was ab96899, DyLight® 488 goat anti-rabbit 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 (asymmetric di methyl R17) antibody - ChIP Grade (ab8284)

This product has been referenced in:
  • McLaughlin N  et al. In situ histone landscape of nephrogenesis. Epigenetics 9:222-35 (2014). IHC-P ; Mouse . Read more (PubMed: 24169366) »
  • Kolodziej S  et al. PADI4 acts as a coactivator of Tal1 by counteracting repressive histone arginine methylation. Nat Commun 5:3995 (2014). ChIP ; Human . Read more (PubMed: 24874575) »

See all 23 Publications for this product

Product Wall

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing (15% resolving gel)
Sample Human Cell lysate - whole cell (Colon cancer cell lines)
Specification Colon cancer cell lines
Username

Mr. Yongchul Lim

Verified customer

Submitted Jun 03 2014

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
Username

Abcam user community

Verified customer

Submitted Feb 21 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Human Tissue lysate - whole (Patient Dermal Fibroblasts)
Specification Patient Dermal Fibroblasts
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Feb 05 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step Serum as blocking agent for 30 minute(s) · Concentration: 3% · Temperature: 37°C
Sample Mouse Cell (Oocyte)
Specification Oocyte
Permeabilization Yes - 0.25% ~0.5% Triton
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Dec 20 2013

Application Western blot
Loading amount 4.5 µg
Gel Running Conditions Reduced Denaturing (12%)
Sample Caenorhabditis elegans Tissue lysate - nuclear (C. elegans whole body)
Specification C. elegans whole body
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted Nov 13 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Rat Cell lysate - whole cell (rat podocyte (kidney cell))
Loading amount 30 µg
Specification rat podocyte (kidney cell)
Gel Running Conditions Reduced Denaturing (14%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 7% · Temperature: 20°C
Username

Mr. Dongil Kim

Verified customer

Submitted May 02 2013

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this...

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Thank you for your call yesterday. I have gotten in touch with my colleagues working in our lab, and unfortunately there is not any of ab8284 left that has passed QC. However, we started working on producing a new batch in July and it takes around 5-6 ...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Cell lysate - whole cell (mouse ES cells)
Loading amount 50 µg
Specification mouse ES cells
Gel Running Conditions Reduced Denaturing (15% SDS-PAGE)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: RT°C
Username

Abcam user community

Verified customer

Submitted Nov 10 2011

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (Hela Cells)
Specification Hela Cells
Fixative Paraformaldehyde
Permeabilization Yes - Yes - 0.25% Triton X-100 in PBS
Blocking step Milk as blocking agent for 11 hour(s) and 0 minute(s) · Concentration: 1% · Temperature: RT°C
Username

Ms. Western,IP ChIP

Verified customer

Submitted Oct 22 2011

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