Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)
Key features and details
- Rabbit polyclonal to Histone H2B (mono methyl K5) - ChIP Grade
- Suitable for: IHC-P, IP, WB, ChIP
- Reacts with: Cow, Human
- Isotype: IgG
Overview
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Product name
Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade
See all Histone H2B primary antibodies -
Description
Rabbit polyclonal to Histone H2B (mono methyl K5) - ChIP Grade -
Host species
Rabbit -
Specificity
Mono methylation of Histone H2B K5 is a putative modification site. ab22512 in ELISA specifically recognises mono-methyl K5 histone H2B peptide but not the corresponding unmodified histone H2B peptide. -
Tested applications
Suitable for: IHC-P, IP, WB, ChIPmore details -
Species reactivity
Reacts with: Cow, Human
Predicted to work with: Mouse, Rat, Chicken, Xenopus laevis
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Immunogen
Synthetic peptide within Human Histone H2B aa 1-100 (mono methyl K5) conjugated to keyhole limpet haemocyanin. The exact sequence is proprietary.
(Peptide available asab13211)
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 or -80°C. Avoid freeze / thaw cycle. -
Storage buffer
pH: 7.40
Preservative: 0.02% Sodium azide
Constituent: PBS
Batches of this product that have a concentration
Concentration information loading...Purity
Immunogen affinity purifiedClonality
PolyclonalIsotype
IgGResearch areas
Associated products
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Compatible Secondaries
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Isotype control
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Recombinant Protein
Applications
Our Abpromise guarantee covers the use of ab12929 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 a concentration of 0.1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol. IP Use at an assay dependent concentration. WB Use a concentration of 1 µg/ml. ChIP Use 2-5 µg for 25 µg of chromatin. Target
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Relevance
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. 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. -
Cellular localization
Nuclear -
Database links
- Entrez Gene: 337874 Human
- Entrez Gene: 54145 Human
- Entrez Gene: 8349 Human
- Entrez Gene: 104021 Mouse
- Entrez Gene: 64647 Rat
- SwissProt: P62808 Cow
- SwissProt: O60814 Human
- SwissProt: P06899 Human
see all -
Alternative names
- GL105 antibody
- H2B GL105 antibody
- H2B histone family member O antibody
see all
Images
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ChIP - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)
Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab12929 (blue), and 20 µl 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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Western blot - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)All lanes : Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929) at 1 µg/ml
Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H2B (mono methyl K5) peptide (ab13211)
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H2B peptide (ab13212)
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 14 kDa -
ChIP - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)This image is courtesy of an anonymous abreview.ab12929 at a 1/600 dilution for ChIP analysis of mouse dorsal skin epidermis whole tissue lysate, incubated for 15 hours at 4°C with ChIP dilution buffer. Cross-linking (X-ChIP) using 1% formaldehyde for 10 minutes.
Detection step: Semiquantitative PCR.Negative control: Rabbit IgG.
Cells untreated. -
Immunoprecipitation - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)Histone H2B (mono methyl K5) was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H2B (mono methyl K5) - ChIP Grade 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 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab12929.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 17kDa: Histone H2B (mono methyl K5). -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)IHC image of Histone H2B (mono methyl K5) staining in human pancreatic adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab12929, 0.1µg/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.
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.
Protocols
Datasheets and documents
References (4)
ab12929 has been referenced in 4 publications.
- Majocchi S et al. Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes. Nucleic Acids Res N/A:N/A (2013). ChIP . PubMed: 24071586
- Santoro SW & Dulac C The activity-dependent histone variant H2BE modulates the life span of olfactory neurons. elife 1:e00070 (2012). ICC/IF ; Mouse . PubMed: 23240083
- Wang Z et al. Combinatorial patterns of histone acetylations and methylations in the human genome. Nat Genet 40:897-903 (2008). WB, ChIP . PubMed: 18552846
- Barski A et al. High-resolution profiling of histone methylations in the human genome. Cell 129:823-37 (2007). PubMed: 17512414
Images
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ChIP - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)
Chromatin was prepared from Hela cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 2 µg of ab12929 (blue), and 20 µl 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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Western blot - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)All lanes : Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929) at 1 µg/ml
Lane 1 : Calf Thymus Histone Preparation Nuclear Lysate (ab121)
Lane 2 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H2B (mono methyl K5) peptide (ab13211)
Lane 3 : Calf Thymus Histone Preparation Nuclear Lysate (ab121) with Human Histone H2B peptide (ab13212)
Lysates/proteins at 20 µg per lane.
Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution
Performed under reducing conditions.
Predicted band size: 14 kDa
-
ChIP - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929) This image is courtesy of an anonymous abreview.ab12929 at a 1/600 dilution for ChIP analysis of mouse dorsal skin epidermis whole tissue lysate, incubated for 15 hours at 4°C with ChIP dilution buffer. Cross-linking (X-ChIP) using 1% formaldehyde for 10 minutes.
Detection step: Semiquantitative PCR.Negative control: Rabbit IgG.
Cells untreated. -
Immunoprecipitation - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)
Histone H2B (mono methyl K5) was immunoprecipitated using 0.5mg Hela whole cell extract, 5µg of Rabbit polyclonal to Histone H2B (mono methyl K5) - ChIP Grade 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 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab12929.
Secondary: Clean blot (HRP conjugate) at 1/1000 dilution.
Band: 17kDa: Histone H2B (mono methyl K5). -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H2B (mono methyl K5) antibody - ChIP Grade (ab12929)
IHC image of Histone H2B (mono methyl K5) staining in human pancreatic adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica Bond™ 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 ab12929, 0.1µg/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.
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.
