Recombinant Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985)
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR18607] to Histone H3 (tri methyl K27) - ChIP Grade
- Suitable for: IHC-P, ChIP, ICC/IF, WB, PepArr, ELISA, ChIP-sequencing
- Reacts with: Mouse, Rat, Human
Related conjugates and formulations
Overview
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Product name
Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade
See all Histone H3 primary antibodies -
Description
Rabbit monoclonal [EPR18607] to Histone H3 (tri methyl K27) - ChIP Grade -
Host species
Rabbit -
Tested applications
Suitable for: IHC-P, ChIP, ICC/IF, WB, PepArr, ELISA, ChIP-sequencingmore details -
Species reactivity
Reacts with: Mouse, Rat, Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HeLa and NIH/3T3 whole cell lysates; Wild type mouse ES whole cell lysate, IHC-P: Human colon, mouse colon and rat kidney tissues. ICC: HeLa cells. ChIP: Chromatin prepared from HeLa cells, Myo-D ChIP primer pair ab269261. ELISA: Histone H3 – unmodified, Histone H3.
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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
Our RabMAb® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb® patents.
Properties
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Form
Liquid -
Storage instructions
Shipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C long term. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.2
Preservative: 0.01% Sodium azide
Constituents: PBS, 40% Glycerol, 0.05% BSA -
Concentration information loading...
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Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR18607 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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ChIP Related Products
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Compatible Secondaries
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Isotype control
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Positive Controls
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Recombinant Protein
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab192985 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 |
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IHC-P | (5) |
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.
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ChIP | (2) |
Use 2 µg for 25 µg of chromatin.
Use Myo-D ChIP primer pair ab269261 as positive control. |
ICC/IF |
1/1000.
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WB | (4) |
1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).
We suggest to use 2% BSA as blocking and antibody dilution buffer. To get stronger band, 1%SDS Hot lysis method is also recommended. |
PepArr |
Use a concentration of 0.1 µg/ml.
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ELISA |
Use a concentration of 0.25 µg/ml.
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ChIP-sequencing |
Use 4µg for 107 cells.
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Notes |
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IHC-P
1/500. Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol. |
ChIP
Use 2 µg for 25 µg of chromatin. Use Myo-D ChIP primer pair ab269261 as positive control. |
ICC/IF
1/1000. |
WB
1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa). We suggest to use 2% BSA as blocking and antibody dilution buffer. To get stronger band, 1%SDS Hot lysis method is also recommended. |
PepArr
Use a concentration of 0.1 µg/ml. |
ELISA
Use a concentration of 0.25 µg/ml. |
ChIP-sequencing
Use 4µg for 107 cells. |
Target
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Function
Core 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 similarities
Belongs to the histone H3 family. -
Developmental stage
Expressed 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 localization
Nucleus. Chromosome. - Information by UniProt
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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
see all -
Alternative names
- H3 histone family member E pseudogene antibody
- H3 histone family, member A antibody
- H3/A antibody
see all
Images
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Chromatin was prepared from HeLa cells. Cells were fixed with 1% formaldehyde for 10 minutes. ChIP was performed with 107 HeLa cells and 4 µg of Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985). ChIP DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 30 million reads.
Additional screenshots of mapped reads can be downloaded here. -
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 ChIP was performed with 25 µg of chromatin, 2 µg of ab192985 (blue), and 20 µl of Anti rabbit IgG sepharose beads. 2 μg of rabbit normal IgG was added to the beads control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).
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Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985)
Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cells from cervix adenocarcinoma) cells labeling Histone H3 (tri methyl K27) with ab192985 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counterstain 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 (red).
The negative controls are as follows:-
-ve control 1: ab192985 at 1/1000 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 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution. -
All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared using 1% SDS hot lysis method
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysate prepared using RIPA lysis method
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/5000 dilution
Predicted band size: 15 kDa
Observed band size: 15 kDa
Exposure time: 3 minutesBlocking/Diluting buffer: 5% NFDM/TBST
For this product, we recommend 1% SDS hot lysis method.
For lysate preparation protocol, please refer to the protocol book in the protocol section or here (downloadable copy).
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985)
Immunohistochemical analysis of paraffin-embedded human colon tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human colon tissue is observed. Counter stained with hematoxylin.
Secondary antibody only control: 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 9.0 before commencing with IHC staining protocol.
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All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution
Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates with 5% NFDM/TBST
Lane 2 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates with 2% BSA/TBST
Lysates/proteins at 15 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) (Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated)
Predicted band size: 15 kDa
Observed band size: 15 kDa
Exposure time: 40 secondsWe recommend to use 2% BSA as blocking and antibody dilution buffer.
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Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution + NIH/3T3 (Mouse embryonic fibroblast cell line) whole cell lysate at 10 µg
Secondary
Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Predicted band size: 15 kDa
Observed band size: 15 kDa
Exposure time: 1 secondBlocking/Dilution buffer: 5% BSA/TBST
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All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution
Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me1 peptide at 5 µg
Lane 3 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me2 peptide at 5 µg
Lane 4 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K4Me3 peptide at 5 µg
Lane 5 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me1 peptide at 5 µg
Lane 6 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me2 peptide at 5 µg
Lane 7 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K9Me3 peptide at 5 µg
Lane 8 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me1 peptide at 5 µg
Lane 9 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me2 peptide at 5 µg
Lane 10 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27Me3 peptide at 5 µg
Lane 11 : HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate with H3K27 unmodified peptide at 5 µg
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution
Developed using the ECL technique.
Predicted band size: 15 kDa
Exposure time: 5 secondsThe antibody is blocked by tri methyl K27 peptide (lane 10) and slightly by di methyl K27 peptide (lane 9, there is 14% cross reactivity with di methyl K27 as determined by ELISA).
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All lanes : Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985) at 1/1000 dilution
Lane 1 : HeLa (Human epithelial cell line from cervix adenocarcinoma)
Lane 2 : EED-/- mouse whole cell lysate
Lane 3 : Wild type mouse ES whole cell lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Developed using the ECL technique.
Predicted band size: 15 kDa
Exposure time: 8 minutes -
Peptide array analysis was performed using ab192985 at a concentration of 0.1 µg/ml, followed by Goat Anti-Rabbit IgG, (H+L), Fluo 647nm conjugated secondary antibody at a 1/50,000 dilution.
ab192985 was tested in Peptide Array against 501 different modified and unmodified histone peptides; each peptide is printed on the array at six concentrations (each in triplicate).
Circle area represents affinity between the antibody and a peptide: all antigen-containing peptides are displayed as red circles, all other peptides as blue circles. The affinity is calculated as area under curve when antibody binding values are plotted against the corresponding peptide concentration. Each circle area is normalized to the peptide with the strongest affinity.
The complete dataset, including full list of all peptides and information on the position of each peptide in the diagram, can be downloaded here.
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ELISA analysis was performed on 1 µg/ml of antigen using ab192985 at a concentration range of 0-0.25 µg/ml, followed by Alkaline Phosphatase-conjugates AffiniPure Goat anti-rabbit IgG (H&L) secondary antibody at a 1/2,500 dilution.
All batches of ab192985 are tested in ELISA against peptides to different Histone H3 modifications. Results show strong binding to Histone H3 - tri methyl K27 immunizing peptide, indicating that this antibody specifically recognizes the Histone H3 - tri methyl K27 modification. Weak binding (14%) was also detected against H3 - di methyl K27 modification.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985)
Immunohistochemical analysis of paraffin-embedded mouse colon tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on mouse colon tissue is observed. Counterstained with hematoxylin.
Secondary antibody only control: 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 9.0 before commencing with IHC staining protocol.
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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (tri methyl K27) antibody [EPR18607] - ChIP Grade (ab192985)
Immunohistochemical analysis of paraffin-embedded rat kidney tissue labeling Histone H3 (tri methyl K27) with ab192985 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on rat kidney tissue is observed. Counterstained with hematoxylin.
Secondary antibody only control: 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 9.0 before commencing with IHC staining protocol.
Protocols
Datasheets and documents
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SDS download
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Datasheet download
Certificate of Compliance
References (42)
ab192985 has been referenced in 42 publications.
- Tavares M et al. JAZF1-SUZ12 dysregulates PRC2 function and gene expression during cell differentiation. Cell Rep 39:110889 (2022). PubMed: 35649353
- Geis FK et al. Two lymphoid cell lines potently silence unintegrated HIV-1 DNAs. Retrovirology 19:16 (2022). PubMed: 35810297
- Liu J et al. lncRNA HOTTIP Recruits EZH2 to Inhibit PTEN Expression and Participates in IM Resistance in Chronic Myeloid Leukemia. Stem Cells Int 2022:9993393 (2022). PubMed: 36117724
- Chen F et al. Different Inhibition of Nrf2 by Two Keap1 Isoforms α and β to Shape Malignant Behaviour of Human Hepatocellular Carcinoma. Int J Mol Sci 23:N/A (2022). PubMed: 36142252
- Huang K & Tang Y SChLAP1 promotes prostate cancer development through interacting with EZH2 to mediate promoter methylation modification of multiple miRNAs of chromosome 5 with a DNMT3a-feedback loop. Cell Death Dis 12:188 (2021). PubMed: 33589600