Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)

Overview

  • Product nameAnti-Histone H3 (mono methyl K4) antibody - ChIP Grade
    See all Histone H3 primary antibodies
  • Description
    Rabbit polyclonal to Histone H3 (mono methyl K4) - ChIP Grade
  • SpecificitySpecific for mono-methylated Lysine 4 of histone H3. Does not recognise di- or tri-methyl Lysine 4 nor methylation at Lysine 9.
  • Tested applicationsIP, Flow Cyt, Electron Microscopy, ICC/IF, ICC, ChIP, WB, CHIPseq, IHC-P, ChIP/Chipmore details
  • Species reactivity
    Reacts with: Mouse, Human, Pig, Saccharomyces cerevisiae, Tetrahymena sp., Xenopus laevis, Fruit fly (Drosophila melanogaster), Plasmodium falciparum, Candida albicans
    Predicted to work with: Cow, Indian Muntjac, Plants, all Mammals
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3, mono methylated at K4.

    (Peptide available as ab1340.)

  • Positive control
    • Calf Thymus Histone Preparation; Hela whole cell extract ICC/IF - HeLa cells

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
  • Clonality Polyclonal
  • IsotypeIgG
  • Research Areas

Applications

Our Abpromise guarantee covers the use of ab8895 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 at an assay dependent concentration.
Flow Cyt 1/100.
Electron Microscopy Use at an assay dependent concentration. PubMed: 20543957
ICC/IF Use at an assay dependent concentration.
IF Use a concentration of 1 µg/ml.

Works better if cells are fixed with methanol.

ICC Use at an assay dependent concentration.
ChIP Use 2 µg for 25 µg of chromatin.
WB 1/500. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (mono methyl K4) peptide (ab1340).
CHIPseq Use at an assay dependent concentration. PubMed: 22196736
IHC-P Use a concentration of 0.5 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ChIP/Chip Use at an assay dependent concentration.

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 (mono methyl K4) antibody - ChIP Grade images

  • ab8895 staining Histone H3 (mono methyl K4) in HeLa cells. The cells were fixed with 100% methanol (5min) and then blocked in 1% BSA/10% normal goat serum/0.3M glycine in 0.1%PBS-Tween for 1h. The cells were then incubated with ab8895 at 1µg/ml and ab7291 (anti beta Tubulin) at 1µg/ml overnight at +4°C, followed by a further incubation at room temperature for 1h with an AlexaFluor®488 Goat anti-Rabbit secondary (ab150081) at 2 μg/ml (shown in green) and AlexaFluor®594 Goat anti-Mouse secondary (ab150120) at 2 μg/ml (shown in pseudo color red). Nuclear DNA was labelled in blue with DAPI.

    Negative controls: 1– Rabbit primary antibody and anti-mouse secondary antibody; 2 – Mouse primary antibody and anti-rabbit secondary antibody. Controls 1 and 2 indicate that there is no unspecific reaction between primary and secondary antibodies used.

  • IHC image of ab8895 staining Histone H3 (mono methyl K4) in human colon formalin fixed paraffin embedded tissue sections, performed on a Leica Bond. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab8895, 0.5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with haematoxylin and mounted with DPX. No primary antibody was used in the negative control (shown on the inset).

     

    For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.

  • Chromatin was prepared from U2OS 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  ab8895 (blue), and 20µl of Protein A/G sepharose beads. No antibody was added to the beads control (yellow). The immunoprecipitated DNA was quantified on the GAPDH and ALDOA (active) and MYO-D (inactive) promoters and over the y-Actin gene (active). Schematic diagram of the y-Actin gene is shown on the top of the figure. Black boxes represent exons and thin lines represent introns. PCR products are depicted as bars under the gene.

  • A: Enlarged region showing the distribution of the H3K4me1 labelling around and within the EC cavities of a murine rod photoreceptor.
    B: Schematic representation of the labelling for the H3K4me1 mark. The bar represents 260 nm in A and 1 µm in B.
  • ab8895 used in Dot Blot at a 1/1000 dilution, 18 hours at 4°C.
    The dot blot was done using 0.1ug of peptide and screened multiple histone tail modifications to ensure no cross reactivity.

    See Abreview

  • Indian muntjac fibroblast cells -  interphase (top left) and prophase (top right and below), stained with:

    Mono Methyl K4 antibody, ab8895, (green)
    DAPI: red, top left and right; blue, below
    Phospho Ser 10 antibody: red (below) and blue (top right)

    The perinuclear and perinucleolar heterochromatin domains do not contain Mono Methyl K4.  The Mono Methyl K4, rather, is distributed as small nuclear foci primarily found between DAPI-intense regions of the nucleus. 

     

  • ab8895 staining mouse embryonic stem cells by flow cytometry. The ES cell colonies were trypsinized and the cells permeabilized before being stained with ab8895 (0.25ug/1.5 x 105 cells). A PE conjugated goat anti-rabbit antibody was used as the secondary.

    See Abreview



  • Performed under reducing conditions.

    Predicted band size : 15 kDa

    ab8895 is specific for mono-methylated Lysine 4 of histone H3 and does not recognize di- or tri-methyl Lysine 4 nor methylation at Lysine 9. This is shown in lane 2 where the activity of the antibody is specifically blocked by the addition of the immunizing peptide (ab1340).

References for Anti-Histone H3 (mono methyl K4) antibody - ChIP Grade (ab8895)

This product has been referenced in:
  • Weedon MN  et al. Recessive mutations in a distal PTF1A enhancer cause isolated pancreatic agenesis. Nat Genet 46:61-4 (2014). ChIP ; Human . Read more (PubMed: 24212882) »
  • Biancolella M  et al. Identification and characterization of functional risk variants for colorectal cancer mapping to chromosome 11q23.1. Hum Mol Genet 23:2198-209 (2014). Read more (PubMed: 24256810) »

See all 161 Publications for this product

Product Wall

Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Na-citrate pH6
Sample Mouse Tissue sections (Liver, P5)
Specification Liver, P5
Permeabilization Yes - Triton 0,05%
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Oct 05 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 20 2014

As both lots are at 1mg/ml concentration, they are BSA free. We add this only to lots below 1mg/ml as a stabilizing agent. Unfortunately, we are unable to do bespoke BSA free orders for such small volumes. I apologize for any inconvenience this may cause.

Thank you for confirming these details and for your cooperation.

Here is our in-house ChIP protocol (the numbering came out a little funny when I pasted it, but it should still be easy to follow):

Scope This standard operation proce...

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Thank you for your inquiry and your patience with this reply while I looked into this with the lab.

For ab8895, the rabbit polyclonal to Histone H3 (mono methyl K4) - ChIP Grade, unfortunately we do not test in mouse so we do not have any mo...

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Hello!

Unfortunately we do not have any smaller sizes available. Becasue of storage capapcity and our large number of products we do not offer any trial sizes, but instead give our guarantee with the Abpromise. Under these terms we can replac...

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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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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Marmoset (common) Tissue sections (adult marmoset testis)
Specification adult marmoset testis
Fixative Formaldehyde
Antigen retrieval step Heat mediated - Buffer/Enzyme Used: Dako Antigen retrieval solution
Permeabilization No
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Zachary Yu-Ching Lin

Verified customer

Submitted Sep 27 2012

For ChIP in general, the Abcam lab always does the IP step overnight (16 hours) at 4C with rotation. For the beads, they prepare a 50:50 mix of protein A/G Sepharose beads (GE Healthcare UK LTD). They typically prepare a new bead slurry every month or ...

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I am sorry to read that this is not working for you. Your protocol must be good if you are getting good results with the K4me1 antibody, but did you try any optimization? Something as simple as adding more antibody may help.

I can send a via...

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