Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)
- Datasheet
- References (11)
- Protocols
Overview
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Product nameAnti-Histone H2B antibody [mAbcam 52484] - ChIP Grade
See all Histone H2B primary antibodies -
DescriptionMouse monoclonal [mAbcam 52484] to Histone H2B - ChIP Grade
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Host speciesMouse
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Tested applicationsSuitable for: IP, Flow Cyt, WB, ICC/IF, ChIP, IHC-P, IHC-Frmore details
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Species reactivityReacts with: Mouse, Rat, Chicken, Human, Drosophila melanogaster, Zebrafish
Predicted to work with: Cow, Xenopus laevis, Caenorhabditis elegans, Orangutan
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Immunogen
Synthetic peptide corresponding to Human Histone H2B aa 100 to the C-terminus (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).
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Positive control
- This antibody gave a positive signal in NIH3T3 and PC12 whole cell lysates and when tested against Histone H2B recombinant protein.
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General notes
This antibody clone is manufactured by Abcam.
If you require this antibody in a particular buffer formulation or a particular conjugate for your experiments, please contact orders@abcam.com or you can find further information here.
Properties
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FormLiquid
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Storage instructionsShipped at 4°C. Store at +4°C short term (1-2 weeks). Upon delivery aliquot. Store at -20°C or -80°C. Avoid freeze / thaw cycle.
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Storage bufferpH: 7.40
Preservative: 0.02% Sodium azide
Constituents: PBS, 6.97% L-Arginine -
Concentration information loading... -
PurityIgG fraction
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ClonalityMonoclonal
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Clone numbermAbcam 52484
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MyelomaSp2/0
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IsotypeIgG1
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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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Immunohistochemistry kits
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Isotype control
Applications
Our Abpromise guarantee covers the use of ab52484 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 |
|---|---|---|
| IP | Use a concentration of 5 µg/ml. | |
| Flow Cyt | Use 1µg for 106 cells. ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody. |
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| WB | Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa). | |
| ICC/IF | Use a concentration of 1 µg/ml. | |
| ChIP | Use 5 µg for µg of chromatin. | |
| IHC-P | Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. | |
| IHC-Fr | Use at an assay dependent concentration. |
Target
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RelevanceCore 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. Subunit structure The nucleosome is a histone octamer containing two molecules each of H2A, H2B, H3 and H4 assembled in one H3-H4 heterotetramer and two H2A-H2B heterodimers. The octamer wraps approximately 147 bp of DNA. Post-translational modification Monoubiquitination at Lys-35 (H2BK34Ub) by the MSL1/MSL2 dimer is required for histone H3 'Lys-4' (H3K4me) and 'Lys-79' (H3K79me) methylation and transcription activation at specific gene loci, such as HOXA9 and MEIS1 loci. Similarly, monoubiquitination at Lys-121 (H2BK120Ub) by the RNF20/40 complex gives a specific tag for epigenetic transcriptional activation and is also prerequisite for histone H3 'Lys-4' and 'Lys-79' methylation. It also functions cooperatively with the FACT dimer to stimulate elongation by RNA polymerase II. H2BK120Ub also acts as a regulator of mRNA splicing: deubiquitination by USP49 is required for efficient cotranscriptional splicing of a large set of exons. Phosphorylation at Ser-37 (H2BS36ph) by AMPK in response to stress promotes transcription. Phosphorylated on Ser-15 (H2BS14ph) by STK4/MST1 during apoptosis; which facilitates apoptotic chromatin condensation. Also phosphorylated on Ser-15 in response to DNA double strand breaks (DSBs), and in correlation with somatic hypermutation and immunoglobulin class-switch recombination. GlcNAcylation at Ser-113 promotes monoubiquitination of Lys-121. It fluctuates in response to extracellular glucose, and associates with transcribed genes. Crotonylation (Kcr) is specifically present in male germ cells and marks testis-specific genes in post-meiotic cells, including X-linked genes that escape sex chromosome inactivation in haploid cells. Crotonylation marks active promoters and enhancers and confers resistance to transcriptional repressors. It is also associated with post-meiotically activated genes on autosomes.
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Cellular localizationNuclear
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Database links
- Entrez Gene: 337874 Human
- Entrez Gene: 54145 Human
- Entrez Gene: 8349 Human
- Entrez Gene: 104021 Mouse
- Entrez Gene: 64647 Rat
- SwissProt: P04255 Caenorhabditis elegans
- SwissProt: P62808 Cow
- SwissProt: P02283 Drosophila melanogaster
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Alternative names
- H2B GL105 antibody
- H2B histone family member O antibody
- H2B histone family member S antibody
see all
Images
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![ChIP - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/ab52484_ 2.jpg)
ChIP - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)Chromatin was prepared from HeLa cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The ChIP was performed with 25µg of chromatin, 5µg of ab52484 (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). Primers and probes are located in the first kb of the transcribed region.
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![Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/ab52484-199738-ab52484icc.jpg)
Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)This image is courtesy of an anonymous Abreviewab52484 staining Histone H2B in Fruit fly (Drosophila melanogaster) embryo cells by ICC/IF (Immunocytochemistry/immunofluorescence). Embryos were washed in 1x PBS + 0.1% Trition, cells were fixed with heat, and blocked with 10% BSA for 12 hours at 4°C. Samples were incubated with primary antibody (1/1000) for 24 hours. An Alexa Fluor®594-conjugated Rabbit anti-mouse IgG polyclonal (1/5000) was used as the secondary antibody.
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![Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/ab52484_1.jpg)
Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)All lanes : Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 5 µg/ml
Lane 1 : Histone H1 recombinant protein.
Lane 2 : Histone H2A recombinant protein.
Lane 3 : Histone H2B recombinant protein.
Lane 4 : Histone H3 recombinant protein.
Lane 5 : Histone H4 recombinant protein.
Lysates/proteins at 0.1 µg per lane.
Secondary
All lanes : Rabbit polyclonal to Mouse IgG - H&L (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? -
![Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/ab52484_Im2.jpg)
Immunocytochemistry/ Immunofluorescence - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)ICC/IF image of ab52484 stained human HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab52484, 1µg/ml) for 1h at room temperature. 1%BSA / 10% normal goat serum / 0.3M glycine was used to block non-specific protein-protein interactions. 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). DAPI was used to stain the cell nuclei (blue).
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![Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/ab52484-ap454714.jpg)
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)IHC image of Histone H2B staining in human breast carcinoma FFPE section, performed on a 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 ab52484, 1µg/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. -
![Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/Histone-H2B-Primary-antibodies-ab52484-1.jpg)
Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)All lanes : Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 5 µg/ml
Lane 1 : Marker
Lane 2 : Zebrafish brain homogenate at 20 µg
Lane 3 : Zebrafish heart homogenate at 10 µg
Lane 4 : Zebrafish liver homogenate at 10 µg
Lane 5 : Zebrafish skeletal muscle homogenate at 10 µg
Lane 6 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg
Secondary
All lanes : Goat polyclonal to Mouse IgG – H&L – Pre-Adsorbed (HRP) at 1/6000 dilution
Developed using the ECL technique.
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: 1 minute -
![Flow Cytometry - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/Histone-H2B-Primary-antibodies-ab52484-5.jpg)
Flow Cytometry - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)Overlay histogram showing HeLa cells stained with ab52484 (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 (ab52484, 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 IgG1 [ICIGG1] (ab91353, 2µg/1x106 cells) used under the same conditions. Acquisition of >5,000 events was performed. -
![Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/Histone-H2B-Primary-antibodies-ab52484-3.jpg)
Western blot - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)All lanes : Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484) at 1 µg/ml
Lane 1 : NIH 3T3 (Mouse embryonic fibroblast cell line) Whole Cell Lysate
Lane 2 : PC12 (Rat adrenal pheochromocytoma cell line) Whole Cell Lysate
Lysates/proteins at 10 µg per lane.
Secondary
All lanes : Goat Anti-Mouse IgG H&L (HRP) preadsorbed (ab97040) at 1/5000 dilution
Developed using the ECL technique.
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: 1 minute -
![Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)](https://www.abcam.com/ps/products/52/ab52484/Images/Histone-H2B-Primary-antibodies-ab52484-6.jpg)
Immunoprecipitation - Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)Histone H2B was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Mouse monoclonal to Histone H2B and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, Hela whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab52484.
Secondary: Protein G-HRP at 1/500 dilution.
Band: 17kDa; Histone H2B; non specific bands - 26kDa: We are unsure as to the identity of this extra band.
References
This product has been referenced in:
- Bui M et al. Internal modifications in the CENP-A nucleosome modulate centromeric dynamics. Epigenetics Chromatin 10:17 (2017). WB ; Human . Read more (PubMed: 28396698) »
- Matilainen O et al. The chromatin remodeling factor ISW-1 integrates organismal responses against nuclear and mitochondrial stress. Nat Commun 8:1818 (2017). Read more (PubMed: 29180639) »
