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Epigenetics and Nuclear Signaling Histones H3 Methylated

Recombinant Anti-Histone H3 (di methyl K36) antibody [EPR16994(2)] - ChIP Class (ab176921)

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Overview

• Product name

• Description

  Rabbit monoclonal [EPR16994(ii)] to Histone H3 (di methyl K36) - Scrap Grade

• Host species

  Rabbit

• Tested applications

  Suitable for: PepArr, ICC/IF, ChIP, IHC-P, WBmore details

• Species reactivity

  Reacts with: Mouse, Rat, Human
  

• Immunogen

  Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.

• Positive control

  • WB: HeLa and NIH/3T3 whole jail cell lysates. IHC-P: Human breadbasket, mouse testis and rat spleen tissues. ICC/IF: HeLa and NIH/3T3 cells. Scrap: Chromatin from HeLa cells.

• General notes

  This product is a recombinant monoclonal antibody, which offers several advantages including:

  • - High batch-to-batch consistency and reproducibility
  • - Improved sensitivity and specificity
  • - Long-term security of supply
  • - Animal-free production

  For more information come across hither.

  Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, delight refer to RabMAb^® patents.

Backdrop

• Course

  Liquid

• Storage instructions

  Shipped at 4°C. Shop at +4°C short term (one-two weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle.

• Storage buffer

  Preservative: 0.01% Sodium azide
  Constituents: 59% PBS, 40% Glycerol (glycerin, glycerine), 0.05% BSA

• Concentration information loading...

• Purity

  Protein A purified

• Clonality

  Monoclonal

• Clone number

  EPR16994(2)

• Isotype

  IgG

• Research areas

Associated products

Applications

The Abpromise guarantee

Our Abpromise guarantee covers the use of ab176921 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be adamant by the end user.

Awarding	Abreviews	Notes
PepArr		Use at an assay dependent concentration.
ICC/IF		1/500.
ChIP		Use 2 µg for 25 µg of chromatin.
IHC-P		1/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH ix.0 before commencing with IHC staining protocol.
WB	(4)	1/5000. Detects a band of approximately 15 kDa (predicted molecular weight: fifteen kDa).

Notes
PepArr Use at an analysis dependent concentration.
ICC/IF 1/500.
Bit Use 2 µg for 25 µg of chromatin.
IHC-P i/250. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
WB i/5000. Detects a ring of approximately 15 kDa (predicted molecular weight: 15 kDa).

Target

• Function

  Core component of nucleosome. Nucleosomes wrap and meaty Dna into chromatin, limiting DNA accessibility to the cellular machineries which require DNA every bit a template. Histones thereby play a central role in transcription regulation, Deoxyribonucleic acid 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 similarities

  Belongs to the histone H3 family.

• Developmental stage

  Expressed during S phase, then expression strongly decreases equally cell division slows downwardly during the process of differentiation.

• Postal service-translational
  modifications

  Acetylation is mostly linked to cistron activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-xix (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
  Citrullination at Arg-ix (H3R8ci) and/or Arg-18 (H3R17ci) past PADI4 impairs methylation and represses transcription.
  Asymmetric dimethylation at Arg-xviii (H3R17me2a) past CARM1 is linked to gene activation. Symmetric dimethylation at Arg-nine (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) past PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the three' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on agile promoters.
  Methylation at Lys-v (H3K4me), Lys-37 (H3K36me) and Lys-eighty (H3K79me) are linked to cistron activation. Methylation at Lys-v (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand intermission (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-eleven (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-ten (H3K9me) and Lys-28 (H3K27me) are enriched in inactive Ten chromosome chromatin.
  Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) past AURKB is crucial for chromosome condensation and prison cell-wheel progression during mitosis and meiosis. In add-on phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase considering 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 every bit c-fos and c-jun. Phosphorylation at Ser-eleven (H3S10ph), which is linked to cistron activation, prevents methylation at Lys-10 (H3K9me) just facilitates acetylation of H3 and H4. Phosphorylation at Ser-eleven (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-eleven (H3S10ph) is as well 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) past JAK2 promotes exclusion of CBX5 (HP1 blastoff) from chromatin.
  Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (Past similarity). Ubiquitinated past 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 localization

  Nucleus. Chromosome.

• Information past UniProt

• Database links

• Alternative names

  • H3 histone family unit fellow member E pseudogene antibody
  • H3 histone family, fellow member A antibody
  • H3/A antibody

  • H31_HUMAN antibiotic
  • H3F3 antibody
  • H3FA antibody
  • Hist1h3a antibody
  • HIST1H3B antibody
  • HIST1H3C antibody
  • HIST1H3D antibody
  • HIST1H3E antibody
  • HIST1H3F antibody
  • HIST1H3G antibody
  • HIST1H3H antibody
  • HIST1H3I antibody
  • HIST1H3J antibody
  • HIST3H3 antibody
  • histone one, H3a antibody
  • Histone cluster 1, H3a antibody
  • Histone H3 3 pseudogene antibody
  • Histone H3.ane antibody
  • Histone H3/a antibody
  • Histone H3/b antibiotic
  • Histone H3/c antibody
  • Histone H3/d antibiotic
  • Histone H3/f antibody
  • Histone H3/h antibody
  • Histone H3/i antibody
  • Histone H3/j antibody
  • Histone H3/1000 antibody
  • Histone H3/l antibody

  come across all

Images

• 

  Flake - Anti-Histone H3 (di methyl K36) antibody [EPR16994(2)] - Scrap Course (ab176921)

  Chromatin was prepared from HeLa (Human epithelial cell line from cervix adenocarcinoma) cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes. The Fleck was performed with 25µg of chromatin, 2µg of ab176921 (red), and 20µl of Anti rabbit IgG sepharose chaplet. 2μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated Deoxyribonucleic acid was quantified past real time PCR (Sybr light-green approach).

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K36) antibody [EPR16994(2)] - ChIP Course (ab176921)

  Immunofluorescent analysis of iv% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling Histone H3 (di methyl K36) with ab176921 at ane/500 dilution, followed past Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/m dilution (green). Confocal image showing nuclear staining on NIH/3T3 cell line. The nuclear counter stain is DAPI (blue).

  Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (crimson).

  The negative controls are every bit follows:
  -ve control 1: ab176921 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution.
  -ve control 2: Anti-alpha Tubulin mouse MAb (ab7291) at 1/chiliad dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• 

  Peptide Array - Anti-Histone H3 (di methyl K36) antibody [EPR16994(2)] - Scrap Grade (ab176921)

  ab176921 was tested in Peptide assortment confronting 501 different modified and unmodified histone peptides; each peptide is printed on the array at 6 concentrations (each in triplicate).

    Circle area represents analogousness betwixt the antibiotic and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blueish circles. The affinity is calculated as surface area under curve when antibiotic bounden values are plotted against the corresponding peptide concentration. Each circle expanse is normalized to the peptide with the strongest affinity.

    The complete dataset, including total list of all peptides and data on the position of each peptide in the diagram, tin can be downloaded here.

• 

  Western absorb - Anti-Histone H3 (di methyl K36) antibiotic [EPR16994(ii)] - ChIP Grade (ab176921)

  All lanes : Anti-Histone H3 (di methyl K36) antibody [EPR16994(two)] - Chip Grade (ab176921) at i/5000 dilution

  Lane i : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
  Lane 2 : NIH/3T3 (Mouse embryonic fibroblast prison cell line) whole cell lysate

  Lysates/proteins at 10 µg per lane.

  Secondary
  All lanes : Caprine animal Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/1000 dilution

  Predicted band size: 15 kDa
  Observed band size: fifteen kDa

  Exposure time: 4 seconds

  Blocking/Dilution buffer: 5% BSA/TBST.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K36) antibiotic [EPR16994(2)] - Flake Grade (ab176921)

  Immunohistochemical analysis of paraffin-embedded homo tummy tissue labeling Histone H3 (di methyl K36) with ab176921 at i/250 dilution, followed by Caprine animal Anti-Rabbit IgG H&L (HRP) (ab97051) at ane/500 dilution. Nuclear staining on man stomach is observed. Counter stained with Hematoxylin.

  Secondary antibody merely command: Used PBS instead of master antibody, secondary antibiotic is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (di methyl K36) antibiotic [EPR16994(2)] - ChIP Grade (ab176921)

  Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling Histone H3 (di methyl K36) with ab176921 at 1/250 dilution, followed past Caprine animal Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse testis is observed. Counter stained with Hematoxylin.

  Secondary antibody only command: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

  Perform heat mediated antigen retrieval with Tris/EDTA buffer pH nine.0 earlier commencing with IHC staining protocol.

• 

  Immunohistochemistry (Formalin/PFA-stock-still alkane-embedded sections) - Anti-Histone H3 (di methyl K36) antibody [EPR16994(2)] - Fleck Grade (ab176921)

  Immunohistochemical analysis of paraffin-embedded rat spleen tissue labeling Histone H3 (di methyl K36) with ab176921 at ane/250 dilution, followed past Goat Anti-Rabbit IgG H&Fifty (HRP) (ab97051) at 1/500 dilution. Nuclear staining on rat spleen is observed. Counter stained with Hematoxylin.

  Secondary antibiotic simply control: Used PBS instead of primary antibiotic, secondary antibody is Goat Anti-Rabbit IgG H&50 (HRP) (ab97051) at 1/500 dilution.

  Perform oestrus mediated antigen retrieval with Tris/EDTA buffer pH 9.0 earlier commencing with IHC staining protocol.

• 

  Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (di methyl K36) antibody [EPR16994(2)] - ChIP Grade (ab176921)

  Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling Histone H3 (di methyl K36) with ab176921 at one/500 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary antibody at ane/thousand dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (bluish).

  Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at one/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/k dilution (reddish).

  The negative controls are as follows:
  -ve control ane: ab176921 at 1/500 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at i/thou dilution.
  -ve command ii: Anti-blastoff Tubulin mouse MAb (ab7291) at 1/grand dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) at i/g dilution.

• 

  Anti-Histone H3 (di methyl K36) antibody [EPR16994(two)] - ChIP Course (ab176921)

Protocols

Datasheets and documents

• Datasheet download

Certificate of Compliance

References (four)

ab176921 has been referenced in 4 publications.

• Xue Wet al. Long non-coding RNAs MACC1-AS1 and FOXD2-AS1 mediate NSD2-induced cisplatin resistance in esophageal squamous cell carcinoma. Mol Ther Nucleic Acids 23:592-602 (2021). PubMed: 33552680
• Bressan RBet al. Regional identity of human neural stem cells determines oncogenic responses to histone H3.3 mutants. Jail cell Stem Cell 28:877-893.e9 (2021). PubMed: 33631116
• Zhou Ret al. Label of H3 methylation in regulating oocyte development in cyprinid fish. Sci People's republic of china Life Sci N/A:Northward/A (2018). PubMed: 30443860
• Streubel Ket al. The H3K36me2 Methyltransferase Nsd1 Demarcates PRC2-Mediated H3K27me2 and H3K27me3 Domains in Embryonic Stalk Cells. Mol Cell 70:371-379.e5 (2018). PubMed: 29606589

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