Anti-Histone H3 (methylated) antibody [2G1-D7] - ChIP Grade (ab89906)

Overview

• Product nameAnti-Histone H3 (methylated) antibody [2G1-D7] - ChIP GradeSee all Histone H3 (methylated) primary antibodies ...
• Description
  Mouse monoclonal [2G1-D7] to Histone H3 (methylated) - ChIP Grade
• Tested applicationsChIP, ICC/IF, WB, IHC-P more details
• Species reactivity
  Reacts with: Cow, Human, Arabidopsis thaliana
  
• Immunogen

  Synthetic peptide of Human Histone H3.

  Read Abcam's proprietary immunogen policy

• Positive controlThis antibody gave a positive signal in the following whole cell lysates: Calf Thymus Histone Nuclear Lysate. This antibody gave a positive result in IHC in the following FFPE tissue: Human normal skin.

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 bufferPreservative: 0.02% Sodium Azide
  Constituents: PBS, pH 7.4
• Concentration information loading...
• PurityIgG fraction
• Clonality Monoclonal
• Clone number2G1-D7
• MyelomaSp2/0-Ag14
• IsotypeIgG1
• Research Areas
  • Epigenetics and Nuclear Signaling
  • ChIP'ing antibodies
  • ChIP'ing antibodies

  • Epigenetics and Nuclear Signaling
  • Histones
  • H3
  • Methylated

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: 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
  • Omim: 601128 Human
  • SwissProt: P59226 Arabidopsis thaliana
  • SwissProt: P68432 Cow
  • SwissProt: Q93081 Human
  • SwissProt: Q16695 Human
  • SwissProt: P68431 Human
  • 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

  see all

• Alternative names
  FLJ92264 antibodyH3 histone antibodyH3 histone family, member A antibody

  H3/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 [2G1-D7] - ChIP Grade images

• 

  Western blot - Histone H3 (mono methyl K9) antibody [2G1-D7] (ab89906)
  All lanes : Anti-Histone H3 (methylated) antibody [2G1-D7] - ChIP Grade (ab89906) 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/ml per lane.
  
  Secondary
  Goat polyclonal to Mouse IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
  developed using the ECL technique
  
  Performed under reducing conditions.
  
  Predicted band size : 15 kDa
  Observed band size : 18 kDa (why is the actual band size different from the predicted?)
  
  
  Exposure time : 1 minute
• 

  ChIP - Histone H3 (mono methyl K9) antibody [2G1-D7] (ab89906)
  Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The ChIP was performed with 25µg of chromatin, 5µg of ab89906 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.
• 

  Immunocytochemistry/ Immunofluorescence - Histone H3 (mono methyl K9) antibody [2G1-D7] (ab89906)
  ICC/IF image of ab89906 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 (ab89906, 1µg/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) Hek293, HepG2, and MCF-7 cells at 1µg/ml, and in 100% Methanol fixed (5 min) HeLa, Hek293, HepG2, MCF-7 cells at 1µg/ml.
• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (mono methyl K9) antibody [2G1-D7] - ChIP Grade (ab89906)

  IHC image of Histone H3 staining in Human normal skin formalin fixed paraffin embedded tissue section, performed on a Leica BondTM system using the standard protocol F. 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 ab89906, 0.2µg/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.

  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.