Anti-Histone H3 (methylated) antibody - ChIP Grade (ab8898)

Overview

• Product nameAnti-Histone H3 (methylated) antibody - ChIP GradeSee all Histone H3 (methylated) primary antibodies ...
• Description
  Rabbit polyclonal to Histone H3 (methylated) - ChIP Grade
• SpecificitySpecific for Histone H3 tri methyl Lysine 9. Shows slight cross-reactivity with tri methyl K27, which shares a similar epitope (please see Western blot image). Does not react with mono or di methylated K9.
• Tested applicationsIHC-Fr, IHC-P, ICC/IF, ChIP, WB, ChIP/Chip, Flow Cyt, CHIPseq more details
• Species reactivity
  Reacts with: Mouse, Rat, Human, Saccharomyces cerevisiae, Fruit fly (Drosophila melanogaster), Indian Muntjac, Cyanidioschyzon merolae
  Predicted to work with: all Mammals
• Immunogen

  Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3, tri methylated at K9.

  Read Abcam's proprietary immunogen policy

  (Peptide available as ab1773.)

• Positive controlCalf Thymus Histone Preparation; Hela whole cell extract
• General notesEvery new batch of this antibody is tested in house in ChIP.

Properties

• FormLiquid
• Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
• 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
  • Epigenetics and Nuclear Signaling
  • Histones
  • H3
  • Methylated

  • Epigenetics and Nuclear Signaling
  • ChIP'ing antibodies
  • ChIP'ing antibodies

Applications

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
  modificationsAcetylation 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.

Target information above from: UniProt accession P68431 The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010) .

Information by UniProt
• Database links
  • Entrez Gene: 8350 Human
  • Entrez Gene: 8351 Human
  • Entrez Gene: 8352 Human
  • Entrez Gene: 8353 Human
  • Entrez Gene: 8354 Human
  • Entrez Gene: 8355 Human
  • Entrez Gene: 8356 Human
  • Entrez Gene: 8357 Human

  • Entrez Gene: 8358 Human
  • Entrez Gene: 8968 Human
  • Entrez Gene: 319152 Mouse
  • Entrez Gene: 319153 Mouse
  • Entrez Gene: 360198 Mouse
  • Entrez Gene: 97908 Mouse
  • Entrez Gene: 100364501 Rat
  • Entrez Gene: 100365669 Rat
  • Entrez Gene: 291159 Rat
  • Entrez Gene: 679994 Rat
  • Entrez Gene: 680511 Rat
  • Entrez Gene: 682330 Rat
  • Omim: 601128 Human
  • SwissProt: P02299 Fruit fly (Drosophila melanogaster)
  • SwissProt: Q93081 Human
  • SwissProt: Q16695 Human
  • SwissProt: P68431 Human
  • SwissProt: P68433 Mouse
  • SwissProt: Q6LED0 Rat
  • Unigene: 132854 Human
  • Unigene: 247813 Human
  • Unigene: 247814 Human
  • Unigene: 248176 Human
  • Unigene: 443021 Human
  • Unigene: 484990 Human
  • Unigene: 532144 Human
  • Unigene: 533292 Human
  • Unigene: 546315 Human
  • Unigene: 586261 Human
  • Unigene: 591778 Human
  • Unigene: 221301 Mouse
  • Unigene: 261657 Mouse
  • Unigene: 377874 Mouse
  • Unigene: 390558 Mouse
  • Unigene: 397328 Mouse
  • Unigene: 138090 Rat

  see all

• Alternative names
  FLJ92264 antibodyH3 histone antibodyH3 histone antibody

  H3 histone family, member A antibodyH3/a antibodyH3/b antibodyH3/c antibodyH3/d antibodyh3/f antibodyH3/h antibodyH3/i antibodyH3/j antibodyH3/k antibodyH3/l antibodyH31_HUMAN antibodyH3F1K antibodyH3F3 antibodyH3F3 antibodyH3FA antibodyH3FB antibodyH3FC antibodyH3FD antibodyH3FF antibodyH3FH antibodyH3FI antibodyH3FJ antibodyH3FK antibodyH3FL antibodyHIST1H3A antibodyHIST1H3B antibodyHIST1H3C antibodyHIST1H3D antibodyHIST1H3E antibodyHIST1H3F antibodyHIST1H3G antibodyHIST1H3H antibodyHIST1H3I antibodyHIST1H3J antibodyHIST3H3 antibodyHIST3H3 antibodyHistone 1, H3a antibodyHistone cluster 1, H3a antibodyHistone cluster 1, H3b antibodyHistone cluster 1, H3c antibodyHistone cluster 1, H3d antibodyHistone cluster 1, H3e antibodyHistone cluster 1, H3f antibodyHistone cluster 1, H3g antibodyHistone cluster 1, H3i antibodyHistone cluster 1, H3j antibodyHistone H 3 antibodyHistone H3.1 antibodyHistone H3.1 antibodyHistone H3/a antibodyHistone H3/b antibodyHistone H3/c antibodyHistone H3/d antibodyHistone H3/f antibodyHistone H3/h antibodyHistone H3/i antibodyHistone H3/j antibodyHistone H3/k antibodyHistone H3/l antibody

  see all

Anti-Histone H3 (methylated) antibody - ChIP Grade images

• 

  ChIP - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)

  Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The  ChIP was performed with 25 µg of chromatin, 2 µg of  ab8898 (blue), and 20 µl 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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

• 

  Western blot - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)
  All lanes : Anti-Histone H3 (methylated) antibody - ChIP Grade (ab8898) at 1 µg/ml
  
  Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
  Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - unmodified (ab7228) at 0.5 µg/ml
  Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K4 (ab1340) at 0.5 µg/ml
  Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K4 (ab7768) at 0.5 µg/ml
  Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K4 (ab1342) at 0.5 µg/ml
  Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 0.5 µg/ml
  Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K9 (ab1772) at 0.5 µg/ml
  Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K9 (ab1773) at 0.5 µg/ml
  Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K27 (ab1780) at 0.5 µg/ml
  Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K27 (ab1781) at 0.5 µg/ml
  Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K27 (ab1782) at 0.5 µg/ml
  
  Lysates/proteins at 0.5 µg per lane.
  
  Secondary
  IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution
  
  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?)
  
• 

  Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)Kirk McManus in the lab of Michael Hendzel, Univeristy of Alberta
  Indian muntjac fibroblast cells stained with anti-Histone H3 tri methyl K9, ab8898, (green, left panel, deconvolution image; red, right panel, epifluorescence image).
  
  The centromeres are enriched in Histone H3 tri methyl K9. There are also additional bands that occur throughout the chromosomes. Note that these images are taken in situ and are imaged under conditions where distinct cytogenetic-like banding patterns have not previously been possible to visualize (e.g., several acetylated antibodies have been reported to be associated with chromosome bands but, although not homogenously distributed along in situ chromosomes, they do not generate distinct banding patterns).
• 

  Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)Kirk McManus in the lab of Michael Hendzel, Univeristy of Alberta
  A 3-D reconstruction of a mouse embryonic fibroblast cell in metaphase stained with anti-Histone H3 tri methyl K9(green, ab8898) and DAPI (red).
• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)This image is courtesy of an Abreview submitted by Dr Patrick Pollard

  ab8898 staining human uterine tumour tissue sections by IHC-P.  Sections were fixed in formaldehyde and subjected to heat mediated antigen retrieval in citrate buffer (pH 6) prior to blocking with 5% serum for 30 minutes at 22°C.  The primary antibody was diluted 1/400 and incubated with the sample for 30 minutes at 22°C.  A HRP-conjugated goat anti-rabbit antibody diluted 1/400, was used as the secondary.

  See Abreview

• 

  ChIP - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)The image is a courtesy of an anonymous abreview.

  X-Chip assay was performed using nuclear lysates prepared from mouse ES cells. Crosslinking was done for 15 minutes in 1% formaldehyde. Primary antibody was incubated first with peptides ab7228, ab1342, ab1782, ab1773, ab1772 and ab1771 in a chip competition assay and then used in chip at 0.0133µg/ µg chromatin (chip sonication buffer) and incubated with sample for 24 hours at 4°C.

  Positive control: ChIP coupled with a peptide competition assay to validate the specificity of the antibody.

  Negative control: Genomic region (chr10:79154149-79155200) with no evidence of H3K9me3. 

  RT-PCR detection method was used.

  Polrmt: PCR primers situated in the coding regions of Polymerase (RNA) mitochondrial (DNA directed).

  Agrn: PCR primers situated in the coding regions of Agrin

• 

  Immunofluorescence - Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)
  These images were kindly submitted by Prof Bryan Turner, University of Birmingham. Undifferentiated Mouse Embryonic Stem cells or cells differentiated for 7 days were incubated with ab8898. The staining is specific for centromeric heterochromatin on metaphase chromosomes.
• 

  Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K9) antibody - ChIP Grade (ab8898)
  ICC/IF image of ab8898 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 (ab8898, 0.1µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 100% methanol fixed (5 min) HepG2 and MCF7 cells at 0.1µg/ml and in 4% PFA fixed (10 min) HeLa, Hek293, HepG2 and MCF7 cells at 0.1ug/ml.
• 

  Immunocytochemistry/ Immunofluorescence-Anti-Histone H3 (tri methyl K9) antibody - ChIP Grade(ab8898)
  ICC/IF image of ab8898 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 (ab8898, 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.
  
  

Protocols

• Chromatin Immunofluorescence