Anti-Histone H2B antibody - ChIP Grade (ab1790)

Overview

  • Product nameAnti-Histone H2B antibody - ChIP Grade
    See all Histone H2B primary antibodies
  • Description
    Rabbit polyclonal to Histone H2B - ChIP Grade
  • SpecificityThis antibody is specific for Histone 2B.
  • Tested applicationsSuitable for: WB, IHC-P, ChIP, ICC/IF, IPmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Cow, Human, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Zebrafish
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 100 to the C-terminus of Human Histone H2B.

    (Peptide available as ab16101.)

  • Positive control
    • Calf Thymus Histone Preparation This antibody gave a positive result when used in the following methanol fixed cell lines: HeLa

Properties

Applications

Our Abpromise guarantee covers the use of ab1790 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
WB Use a concentration of 0.1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 14 kDa).
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.
ChIP Use a concentration of 2 - 3 µg/ml.
ICC/IF Use a concentration of 0.5 µg/ml.
IP Use a concentration of 5 µg/ml.

Target

  • 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.
  • Cellular localizationNuclear
  • Database links
  • Alternative names
    • H2B GL105 antibody
    • H2B histone family member O antibody
    • H2B histone family member S antibody
    • H2B.1 antibody
    • H2B.1 B antibody
    • H2B.b antibody
    • H2B.c antibody
    • H2B.d antibody
    • H2B.e antibody
    • H2B.f antibody
    • H2B.j antibody
    • H2B.q antibody
    • H2B/b antibody
    • H2B/c antibody
    • H2B/d antibody
    • H2B/e antibody
    • H2B/f antibody
    • H2B/j antibody
    • H2B/o antibody
    • H2B/q antibody
    • H2BFB antibody
    • H2BFC antibody
    • H2BFD antibody
    • H2BFE antibody
    • H2BFF antibody
    • H2BFJ antibody
    • H2BFO antibody
    • H2BFQ antibody
    • H2BFS antibody
    • HIRIP2 antibody
    • HIST1H2BB antibody
    • HIST1H2BD antibody
    • HIST1H2BH antibody
    • HIST1H2BL antibody
    • HIST1H2BM antibody
    • HIST1H2BN antibody
    • HIST2H2BE antibody
    • Histone H2B antibody
    • Histone H2B type 1 B antibody
    • Histone H2B type 1 D antibody
    • Histone H2B type 1 H antibody
    • Histone H2B type 1 L antibody
    • Histone H2B type 1 M antibody
    • Histone H2B type 1 N antibody
    • Histone H2B type 2 E antibody
    • histone protein antibody
    see all

Anti-Histone H2B antibody - ChIP Grade images

  • Chromatin was prepared from U2OS cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10min. The  ChIP was performed with 25µg of chromatin, 2µg of ab1790 (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 for active and inactive loci, Sybr green approach for heterochromatic loci). Primers and probes are located in the first kb of the transcribed region.

  • Histone H2B - ChIP Grade was immunoprecipitated using 0.5mg HeLa whole cell extract, 5µg of Rabbit polyclonal to 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 ab1790.

    Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

    Band: 14kDa; Histone H2B - ChIP Grade

  • ICC/IF image of ab1790 stained HeLa cells. The cells were 100% methanol fixed (5 min) then permeabilised using 0.1% PBS-Triton and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to further permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab1790 at 0.1µg/ml overnight at +4°C. The secondary antibody (pseudo-colored green) was Alexa Fluor® 488 goat anti- rabbit (ab150081) IgG (H+L) preadsorbed, used at a 1/1000 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (pseudo-colored red) at a 1/200 dilution for 1h at room temperature. DAPI was used to stain the cell nuclei (pseudo-colored blue) at a concentration of 1.43µM for 1hour at room temperature.

  • Chromatin from Xenopus laevis oocytes was prepared according to the Abcam X-ChIP protocol. Oocytes were fixed with formaldehyde for 10 min. The ChIP was performed with 25 µg of chromatin, 3 µg of ab1790 (anti-H2B, light blue) and 3 µg of ab1791 (anti-H3, dark blue), and 20 µl of Protein A/G sepharose beads. A non-specific antibody was used as a control (yellow). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach).



  • Performed under reducing conditions.

    Predicted band size : 14 kDa
  • All lanes : Anti-Histone H2B antibody - ChIP Grade (ab1790)

    Lane 1 : Hela Histone prep
    Lane 2 : Hela whole cell lysate
    Lane 3 : S. cerevisiae whole cell lysate


    Performed under reducing conditions.

    Predicted band size : 14 kDa

  • developed using the ECL technique

    Performed under reducing conditions.

    Predicted band size : 14 kDa
    Observed band size : 17 kDa (why is the actual band size different from the predicted?)


    Exposure time : 2 seconds

    This image is courtesy of an Abreview submitted by Dr Igor Landais

    ab1790 at 1/3000 detecting Histone H2B from Xenopus laevis (S phase egg extracts - whole cell lysates 60ug per lane) by Western Blot. The egg extracts were fractionated using a gel filtration column and every other fraction (4 - 26) was loaded onto a 8-16% gel. The input corresponds to 1ul of crude extract. In this experiment an HRP conjugated donkey anti-rabbit antibody was used as the secondary.

    See Abreview

  • HeLa cells were fixed in 100% methanol for 6 minutes at -20°C. The cells were washed 3 times in PBS then incubated with ab1790 (0.5µg/ml) for 1 hour at room temperature. The panel of images shows the cells stained with ab1790 (green) and counterstained with DAPI (blue). 100x magnification.

  • 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 ab1790, 1µg/ml, for 8 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.

References for Anti-Histone H2B antibody - ChIP Grade (ab1790)

This product has been referenced in:
  • Rønningen T  et al. Pre-patterning of differentiation-driven nuclear lamin A/C-associated chromatin domains by GlcNAcylated histone H2B. Genome Res N/A:N/A (2015). ChIP ; Human . Read more (PubMed: 26359231) »
  • Milev MP  et al. TRAMM/TrappC12 plays a role in chromosome congression, kinetochore stability, and CENP-E recruitment. J Cell Biol 209:221-34 (2015). WB, ICC/IF ; Human . Read more (PubMed: 25918224) »

See all 58 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa cells)
Specification HeLa cells
Fixative Formaldehyde
Permeabilization Yes - 0.1 % TritonX-100 for 5 minutes
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 2% · Temperature: 25°C
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Abcam user community

Verified customer

Submitted Jul 13 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Human Cell lysate - nuclear (HeLa cell line)
Total protein in input 5e+006 cells
Specification HeLa cell line
Immuno-precipitation step Protein A
Username

Abcam user community

Verified customer

Submitted Mar 28 2011

Application Western blot
Sample Human Cell lysate - whole cell (HeLa)
Gel Running Conditions Reduced Denaturing
Loading amount 10 µg
Specification HeLa
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
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Submitted Jul 01 2016

Application Immunohistochemistry (Resin sections)
Sample Astatotilapia burtoni Tissue sections (Brain Preoptic area LRWhite embedded)
Specification Brain Preoptic area LRWhite embedded
Username

Dr. Sebastian Alvarado

Verified customer

Submitted May 03 2016

Application Western blot
Loading amount 0.5 µg
Gel Running Conditions Reduced Denaturing (18% gel)
Sample Human Recombinant protein (Recombinant octamers & purified K-562 cell histone)
Specification Recombinant octamers & purified K-562 cell histone
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 3% · Temperature: 25°C
Username

Dr. Ragnhild Eskeland

Verified customer

Submitted Mar 04 2015

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Xenopus laevis Cell lysate - nuclear (Egg extract (total and chromatin))
Specification Egg extract (total and chromatin)
Blocking step Milk as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: RT°C
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Submitted Sep 03 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Xenopus laevis Cell lysate - whole cell (Erythrocytes)
Loading amount 50000 cells
Specification Erythrocytes
Gel Running Conditions Reduced Denaturing (12%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Submitted Apr 01 2013

The immunogen sequence used to raise the anti-Histone H2B antibodies (ab1790 and ab52484) is proprietary information. However, I can say that the immunogen is a synthetic peptide taken from within the residues 100 to the C-terminal of human Histone H2B...

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Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunoprecipitation
Sample Xenopus laevis Cell lysate - other (Egg)
Total protein in input 625 µg
Specification Egg
Immuno-precipitation step Other - Dynabeads
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Abcam user community

Verified customer

Submitted Feb 27 2013

Both of these antibodies are IgG antibodies. Unfortunately, we have not determined the subclass(es) of IgG present but I would expect there to be a mix of subclasses with possibly one predominating but this could vary depending on the immune response o...

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