Recombinant Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)
![ChIP - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)](https://www.abcam.com/ps/products/273/ab273635/Images/ab273635-396487-anti-histone-h3-3-mutated-g34r-antibody-epr235199-chip.jpg "ChIP - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)")
Key features and details
- Produced recombinantly (animal-free) for high batch-to-batch consistency and long term security of supply
- Rabbit monoclonal [EPR23519-91] to Histone H3.3 (mutated G34R) - ChIP Grade – BSA and Azide free
- Suitable for: IHC-P, Dot blot, WB, ChIP, Flow Cyt (Intra), IP
- Reacts with: Human
Related conjugates and formulations
Overview
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Product name
Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free
See all Histone H3.3 primary antibodies -
Description
Rabbit monoclonal [EPR23519-91] to Histone H3.3 (mutated G34R) - ChIP Grade – BSA and Azide free -
Host species
Rabbit -
Tested applications
Suitable for: IHC-P, Dot blot, WB, ChIP, Flow Cyt (Intra), IPmore details
Unsuitable for: ICC/IF -
Species reactivity
Reacts with: Human -
Immunogen
Synthetic peptide. This information is proprietary to Abcam and/or its suppliers.
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Positive control
- WB: HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®, whole cell lysate. IHC-P: Human giant tumor of bone tissue. Flow Cyt (intra): HEK-293T cells transfected with myc-tagged Histone H3.3 G34R expression vector. IP: HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag® whole cell lysate. ChIP: Chromatin prepared from HEK-293T transfected with Histone H3.3 (mutated G34 R).
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General notes
ab273635 is the carrier-free version of ab254402.
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. The carrier-free buffer and high concentration allow for increased conjugation efficiency.
This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.
Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with <1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.
This product is compatible with the Maxpar® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar® is a trademark of Fluidigm Canada Inc.
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. Do Not Freeze. -
Storage buffer
pH: 7.2
Constituent: PBS -
Carrier free
Yes -
Concentration information loading... -
Purity
Protein A purified -
Clonality
Monoclonal -
Clone number
EPR23519-91 -
Isotype
IgG -
Research areas
Associated products
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Alternative Versions
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Compatible Secondaries
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Isotype control
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Related Products
Applications
The Abpromise guarantee
Our Abpromise guarantee covers the use of ab273635 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 |
|---|---|---|
| IHC-P |
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
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| Dot blot |
Use at an assay dependent concentration.
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| WB |
Use at an assay dependent concentration. Detects a band of approximately 20 kDa (predicted molecular weight: 15 kDa).
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| ChIP |
Use at an assay dependent concentration.
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| Flow Cyt (Intra) |
Use at an assay dependent concentration.
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| IP |
Use at an assay dependent concentration.
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| Notes |
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IHC-P
Use at an assay dependent concentration. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol. |
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Dot blot
Use at an assay dependent concentration. |
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WB
Use at an assay dependent concentration. Detects a band of approximately 20 kDa (predicted molecular weight: 15 kDa). |
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ChIP
Use at an assay dependent concentration. |
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Flow Cyt (Intra)
Use at an assay dependent concentration. |
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IP
Use at an assay dependent concentration. |
Target
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Function
Variant histone H3 which replaces conventional H3 in a wide range of nucleosomes in active genes. Constitutes the predominant form of histone H3 in non-dividing cells and is incorporated into chromatin independently of DNA synthesis. Deposited at sites of nucleosomal displacement throughout transcribed genes, suggesting that it represents an epigenetic imprint of transcriptionally active chromatin. 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 throughout the cell cycle independently of DNA synthesis. -
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.
Specifically enriched in modifications associated with active chromatin such as methylation at Lys-5 (H3K4me), Lys-37 and Lys-80. 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), which are linked to gene repression, are underrepresented. 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. Phosphorylation on Ser-32 (H3S31ph) is specific to regions bordering centromeres in metaphase chromosomes.
Ubiquitinated. Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination. -
Cellular localization
Nucleus. Chromosome. - Information by UniProt
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Database links
- Entrez Gene: 3020 Human
- Entrez Gene: 3021 Human
- Omim: 601058 Human
- SwissProt: P84243 Human
- Unigene: 180877 Human
- Unigene: 533624 Human
- Unigene: 726012 Human
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Alternative names
- H3 histone family 3A antibody
- H3 histone family 3B antibody
- H3 histone, family 3B (H3.3B) antibody
see all
Images
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ChIP - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)
Chromatin was prepared from 293T transfacted with Histone H3.3(mutated G34 R) and 293T transfacted with Histone H3.3(WT)
cells according to the Abcam X-ChIP protocol*. Cells were fixed with formaldehyde for 10min.
The ChIP was performed with 25 µg of chromatin, 2 µg of ab254402 (red), or 2 µg of rabbit normal IgG ab172730 (gray) and 25 µl of Protein A/G Dynabeads. The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci).*https://www.abcam.com/resources?keywords=X%20ChIP%20protocol
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254402).
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![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)](https://www.abcam.com/ps/products/273/ab273635/Images/ab273635-1-anti-histone-h33-mutated-g34-r-antibody-epr2351991-immunohistochemistry-human-giant-cell-tumor-of-bone.jpg)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)Immunohistochemical analysis of paraffin-embedded Human giant cell tumor of bone tissue labeling Histone H3.3 (mutated G34 R) with ab254402 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human giant cell tumor of bone (PMID: 29241742). The section was incubated with ab254402 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254402).
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![Western blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)](https://www.abcam.com/ps/products/273/ab273635/Images/ab273635-396397-anti-histone-h33-mutated-g34-r-antibody-epr2351991-western-blot-hek293t.jpg)
Western blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)All lanes : Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade (ab254402) at 1/1000 dilution
Lane 1 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R expression vector containi a myc-His-tag®, whole cell lysate
Lane 2 : HEK-293T transfected with Histone H3.3 (WT) expression vector containi a myc-His-tag®, whole cell lysate
Lane 3 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34V expression vector containi a myc-His-tag®, whole cell lysate
Lane 4 : HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34W expression vector containi a myc-His-tag®, whole cell lysate
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution
Predicted band size: 15 kDa
Observed band size: 20 kDa why is the actual band size different from the predicted?Blocking and diluting buffer and concentration: 5% NFDM/TBST.
Exposure time: 26 seconds .
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254402).
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![Flow Cytometry (Intracellular) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)](https://www.abcam.com/ps/products/273/ab273635/Images/ab273635-3-anti-histone-h33-mutated-g34-r-antibody-epr2351991-flow-cytometry-293t.jpg)
Flow Cytometry (Intracellular) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)Intracellular flow cytometric analysis of 4% paraformaldehyde fixed, 90% methanol permeabilized HEK-293T (Human embryonic kidney epithelial cell) (transfected with myc-tagged Histone H3.3 WT expression vector) (Left) or myc-tagged Histone H3.3 G34R expression vector (Right) cells labelling Histone H3.3 (mutated G34 R) with ab254402 at 1/5000 dilution (0.01ug) (Both panels). A Goat anti rabbit IgG (Alexa Fluor®488, ab150077) at 1/2000 dilution was used as the secondary antibody.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254402).
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![Immunoprecipitation - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)](https://www.abcam.com/ps/products/273/ab273635/Images/ab273635-4-anti-histone-h33-mutated-g34-r-antibody-epr2351991-immunoprecipitation-hek293t.jpg)
Immunoprecipitation - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)Histone H3.3 (mutated G34 R) was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney) (transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®) whole cell lysate with ab254402 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab254402 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.
Lane 1: HEK-293T (human embryonic kidney) transfected with Histone H3.3 G34R expression vector containing a myc-His-tag®, whole cell lysate 10 ug
Lane 2: ab254402 IP in HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag® whole cell lysate
Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab254402 in HEK-293T transfected with Histone H3.3 G34R expression vector containing a myc-His-tag® whole cell lysate
Blocking and dilution buffer and concentration: 5% NFDM/TBST.
Exposure time: 41 seconds.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254402).
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![Dot Blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)](https://www.abcam.com/ps/products/273/ab273635/Images/ab273635-396488-anti-histone-h3-3-mutated-g34r-antibody-epr235199-dot-blot.jpg)
Dot Blot - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)Dot blot analysis of Histone H3.3 (mutated G34 R) labeled with ab254402 at 1/1000 dilution.
Lane 1: Histone H3.3 H3G34R peptide (aa28-40).
Lane 2: Histone H3.3 H3G34R peptide (aa26-36).
Lane 3: Histone H3.3 WT peptide (aa26-40).Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/100000 dilution was used as secondary antibody.
Blocking and dilution buffer: 5% NFDM/TBST.
Exposure time: 3 minutes.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254402).
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![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)](https://www.abcam.com/ps/products/273/ab273635/Images/ab273635-2-anti-histone-h33-mutated-g34-r-antibody-epr2351991-immunohistochemistry-human-testis.jpg)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)Immunohistochemical analysis of paraffin-embedded Human testis tissue labeling Histone H3.3 (mutated G34 R) with ab254402 at 1/2000 dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: no staining on human testis. The section was incubated with ab254402 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument. Counterstained with Hematoxylin.
Secondary antibody only control: Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).
Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.
This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab254402).
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![Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)](https://www.abcam.com/ps/products/273/ab273635/Images/ab273635-8-benefits-of-recombinant-antibodies.png)
Anti-Histone H3.3 (mutated G34R) antibody [EPR23519-91] - ChIP Grade – BSA and Azide free (ab273635)
Protocols
To our knowledge, customised protocols are not required for this product. Please try the standard protocols listed below and let us know how you get on.
Datasheets and documents
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Datasheet download
Certificate of Compliance
References (0)
ab273635 has not yet been referenced specifically in any publications.
