Anti-Histone H3 (methylated) antibody [mAbcam1012] - ChIP Grade (ab1012)

Overview

• Product nameAnti-Histone H3 (methylated) antibody [mAbcam1012] - ChIP GradeSee all Histone H3 (methylated) primary antibodies ...
• Description
  Mouse monoclonal [mAbcam1012] to Histone H3 (methylated) - ChIP Grade
• Specificityab1012 is strongly blocked in Western blotting on histones by tri methyl K4, partially by di methyl K4 and very weakly by mono methyl K4 peptides. It is not blocked by non-modified peptides. By ELISA the antibody binds to the tri methyl K4 peptide and partially to di and mono methyl K4 peptides. It does not bind to unmodified, mono, di or tri methyl K9 or di or tri methyl K27 peptides. Not suitable for blocking with milk in Western blot (see Application notes).
• Tested applicationsChIP, Flow Cyt, ICC/IF, IHC-Fr, WB, ICC more details
• Species reactivity
  Reacts with: Mouse, Rat, Cow, Human, Zebrafish, Rice
  Predicted to work with: Sheep, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, all Mammals
• Immunogen

  Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3.

  Read Abcam's proprietary immunogen policy

  (Peptide available as ab1342.)

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: 7.4
  Preservative: 0.02% Sodium azide
  Constituent: PBS
• Concentration information loading...
• PurityIgG fraction
• Clonality Monoclonal
• Clone numbermAbcam1012
• IsotypeIgG2b
• Light chain typekappa
• 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: 176359 Caenorhabditis elegans
  • Entrez Gene: 326601 Cow
  • 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: P59226 Arabidopsis thaliana
  • SwissProt: Q10453 Caenorhabditis elegans
  • SwissProt: P08898 Caenorhabditis elegans
  • SwissProt: P68432 Cow
  • 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 antibodyH3FA antibodyH3FB antibodyH3FC antibodyH3FD antibodyH3FF antibodyH3FH antibodyH3FI antibodyH3FJ antibodyH3FK antibodyH3FL antibodyHIST1H3A antibodyHIST1H3B antibodyHIST1H3C antibodyHIST1H3D antibodyHIST1H3E antibodyHIST1H3F antibodyHIST1H3G antibodyHIST1H3H antibodyHIST1H3I antibodyHIST1H3J 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 [mAbcam1012] - ChIP Grade images

• 

  Immunocytochemistry - Histone H3 (tri methyl K4) antibody [mAbcam 1012] (ab1012)Darin McDonald, Cross Cancer Institute
  SK-N-SK cells were fixed in 4% paraformaldehdye, permeabilized in 0.5% Triton X-100 and incubated with ab1012 (1/100). The antibody clearly stains the nucleus (red). The cells were counterstained with DAPI (blue). 100x magnification.
• 

  ChIP - Histone H3 (tri methyl K4) antibody [mAbcam 1012] - ChIP Grade (ab1012)

  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 25 µg of chromatin, 5 µg of  ab1012 (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). Primers and probes are located in the first kb of the transcribed region.

• 

  Immunocytochemistry/ Immunofluorescence - Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012)
  ICC/IF image of ab1012 stained HeLa cells. The cells were 4% PFA fixed (10 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 (ab1012, 5µg/ml) overnight at +4°C. The secondary antibody (green) was Alexa Fluor® 488 goat anti-mouse 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 4% PFA fixed (10 min) HepG2, Hek293 and MCF7 cells at 5µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 5µg/ml.
• 

  Western blot - Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012)
  Lane 1 : Anti-Histone H3 (methylated) antibody [mAbcam1012] - ChIP Grade (ab1012) at 1 µg/ml (Blocked in 5% BSA)
  Lane 2 : Anti-Histone H3 (methylated) antibody [mAbcam1012] - ChIP Grade (ab1012) at 1 µg/ml (Blocked in 5% MILK)
  
  Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate
  Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate
  
  Lysates/proteins at 0.5 µg per lane.
  
  Secondary
  Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
  
  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 : 10 minutes
• 

  Western blot - Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012)
  All lanes : Anti-Histone H3 (methylated) antibody [mAbcam1012] - ChIP Grade (ab1012) at 2 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 0.25 µg/ml
  Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K4 (ab1340) at 0.25 µg/ml
  Lane 4 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K4 (ab7768) at 0.25 µg/ml
  Lane 5 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K4 (ab1342) at 0.25 µg/ml
  Lane 6 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K9 (ab1771) at 0.25 µg/ml
  Lane 7 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K9 (ab1772) at 0.25 µg/ml
  Lane 8 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K9 (ab1773) at 0.25 µg/ml
  Lane 9 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - mono methyl K27 (ab1780) at 0.25 µg/ml
  Lane 10 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - di methyl K27 (ab1781) at 0.25 µg/ml
  Lane 11 : Calf Thymus Histone Preparation Nuclear Lysate with Histone H3 peptide - tri methyl K27 (ab1782) at 0.25 µg/ml
  
  Lysates/proteins at 0.5 µg per lane.
  
  Secondary
  Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
  
  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 : 16 minutes
• 

  ELISA - Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade (ab1012)

  ELISA using ab1012 at varying antibody concentrations.

  Curve_SPL4 indicates binding to the tri methyl K4 peptide ab1342. Curve SPL3 indicates partial binding to the di methyl K4 peptide ab7768. There is very weak cross-reactivity with the mono methyl K4 peptide ab1340 (Curve_SPL2).

  Binding to the following peptides was not seen: SPL1 unmodified Histone H3, SPL5 Histone H3 mono methyl K9, SPL6 Histone H3 di methyl K9, SPL7 Histone H3 tri methyl K9, SPL8 Histone H3 mono methyl K27, SPL9 Histone H3 di methyl K27, SPL10 Histone H3 tri methyl K27.

• 

  Flow Cytometry-Anti-Histone H3 (tri methyl K4) antibody [mAbcam1012] - ChIP Grade(ab1012)
  Overlay histogram showing HeLa cells stained with ab1012 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab1012, 1µg/1x10⁶ cells) for 30 min at 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG2b [PLPV219] (ab91366, 2µg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed.