Overview

  • Product name

    Anti-Histone H2B antibody - ChIP Grade
    See all Histone H2B primary antibodies
  • Description

    Rabbit polyclonal to Histone H2B - ChIP Grade
  • Host species

    Rabbit
  • Specificity

    This antibody is specific for Histone 2B.
  • Tested applications

    Suitable 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 corresponding to Human Histone H2B aa 100 to the C-terminus conjugated to keyhole limpet haemocyanin.
    (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

  • 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

    Preservative: 0.02% Sodium Azide
    Constituents: 1% BSA, PBS, pH 7.4
  • Concentration information loading...
  • Purity

    Immunogen affinity purified
  • Clonality

    Polyclonal
  • Isotype

    IgG
  • Light chain type

    unknown
  • Research areas

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).Can be blocked with Human Histone H2B peptide (ab16101).
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

  • 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

  • Alternative names

    • GL105 antibody
    • 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
    • H2BGL105 antibody
    • H2BQ antibody
    • HIRIP2 antibody
    • HIST1H2BB antibody
    • HIST1H2BD antibody
    • HIST1H2BH antibody
    • HIST1H2BL antibody
    • HIST1H2BM antibody
    • HIST1H2BN antibody
    • HIST2H2BE antibody
    • histone cluster 2 antibody
    • Histone H2B antibody
    • histone H2B GL105 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 H2B.q antibody
    • histone protein antibody
    see all

Images

  • 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

  • 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.

  • 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.

  • 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

  • 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.
  • 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).

  • All lanes : Anti-Histone H2B antibody - ChIP Grade (ab1790) at 0.1 µg/ml

    Lane 1 : Calf thymus histone prep
    Lane 2 : Calf thymus histone prep with Human Histone H2B peptide (ab16101) at 1 µg/ml

    Lysates/proteins at 20 µg per lane.

    Secondary
    All lanes : Alexa fluor Goat polyclonal anti-Rabbit IgG at 1/10000 dilution

    Performed under reducing conditions.

    Predicted band size: 14 kDa

  • 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.

  • 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

References

This product has been referenced in:

  • Yeo D  et al. Intensified mitophagy in skeletal muscle with aging is downregulated by PGC-1alpha overexpression in vivo. Free Radic Biol Med 130:361-368 (2019). Read more (PubMed: 30395971) »
  • Libetti D  et al. NF-YA enters cells through cell penetrating peptides. Biochim Biophys Acta Mol Cell Res 1866:430-440 (2019). Read more (PubMed: 30296497) »
See all 145 Publications for this product

Customer reviews and Q&As

1-10 of 20 Q&A

Answer

Thank you for confirming these details and for your cooperation. The details provided enable us to closely monitor the quality of our products.

I am sorry this product did not perform as stated on the datasheet and for the inconvenience this has caused. As requested, I have issued a free of charge replacement with the order number 1192133.

To check the status of the order please contact our Customer Service team and reference this number.

Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

I wish you the best of luck with your research.

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Answer

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. The immunogen used to raise these two antibodies is the same.
If you could explain a little further why you need this information I may be able to help further.

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Question
Answer

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 of the rabbit for ab1790 and the mouse for ab11503.

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Answer

Thank you for contacting us.

The stock for lot 632626 is sold out we cannot provide this. We however have new lot in stock, would you like try a new vial?

Could you also provide the purchase order number so that we know where to ship the antibody?

Thanks, looking forward to hearing from you soon.

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Answer

Thank you for contacting us.

I am sorry to hear you are experiencing difficulties with one of our products. We take product complaints very seriously, and investigate every product that we feel may not be performing correctly.

I am attaching our questionnaire so that we can gather further information regarding the samples tested and the protocol used. Once we receive the completed questionnaire, we will look at the protocol and see if there are any suggestions we can make that may improve the results. This information will also allow us to investigate this case internally and initiate additional testing where necessary. If the product was purchased in the last six months and is being used according to our Abpromise, we would be happy to replace or refund the antibody.

I look forward to receiving your reply.

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Answer

Thank you for contacting us.

Currently our Rabbit polyclonal to Histone H2B - ChIP Grade (ab1790) lot # GR67993-1 has a concentration of 1mg/ml. There are other lots available which have a lower concentration so be sure to request this specific lot.

All lots of Rabbit polyclonal to Histone H3 - ChIP Grade (ab1791) currently in stock have a concentration of 1mg/ml.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.



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https://www.abcam.com/abreviews

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Answer

Thank you for contacting us.

These products are sold in liquid form and should not require any reconstitution. When using in your particular assay please dilute in the appropriate buffer for that assay.

I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

Use our products? Submit an Abreview. Earn rewards!
https://www.abcam.com/abreviews

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Answer

Thank you for your enquiry and your interest in our products.
I can confirm that ab1790: Anti-Histone H2B antibody - ChIP Grade has been specifically designed to recognize H2B. The specificity of this antibody has been tested in blocking experiments (WB) and ChIP. Please take a look at the datasheet for further information about this product.
If you need any further assistance in the future, please do not hesitate to contact me.

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Answer

Thank your for your interest in ab1790. The immunogen is identical to the homologous yeast sequence at 14 out of 18 amino acids. S. cerevisiae is listed as a reactive species based on a reference that used the antibody to detect yeast H2B: Camahort R et al. Scm3 is essential to recruit the histone h3 variant cse4 to centromeres and to maintain a functional kinetochore. Mol Cell 26:853-65 (2007). PubMed: 17569568. We did receive a negative review in 2006 from a customer who was unable to detect yeast H2B, and it is believed there may be lot-to-lot variation with respect to yeast reactivity. The species is still covered by our guarantee: if it fails to detect yeast H2B in an application listed on the datasheet, we will offer a replacement, credit, or refund if we are unable to suggest protocol improvements. I hope this helps. If you have any questions, please contact us.

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Answer

Thank you for your inquiry and I am sorry to hear that you are having trouble with ab1790 and ab9051. Thank you for providing your protocol information and testing images. Having reviewed the information provided, I would like to offer some suggestions to improve the results. How much protein are you loading per lane? We would recommend to load at least 30 ug/lane. Are these run in reduced and denatured conditions? Instead of doing the block for 45 mins, primary, rinse, block for 45 mins, secondary, rinse, and then detection, you should omit the second blocking step as this is not necessary and may be preventing the detection of the band of interest and incubate the primary overnight instead of 2 hrs. You should also try blocking with BSA instead of milk, since this is what we've tested in-house. I would recommend the following protocol: After transfer, rinse the membrane. Block the membrane with 3% BSA for 1 hr at RT on a shaker. Wash 3 x 5 mins with TBST. Incubate primary 1/1000 overnight at 4C in 3% BSA + TBST on a shaker. Wash 3 x 5 mins with TBST. Incubate anti-rabbit secondary 1/5000 for 1 hr at RT in 3% BSA + TBST. As for Anti-Histone H4 (mono methyl K20) antibody - ChIP Grade (ab9051), we haven't tested this antibody with rat samples. We "predict" it to cross-react due to high sequence homology, but we haven't actually tested nor can we guarantee that this antibody will work in rat. You may want to run a human or mouse positive control. I hope this information helps and please let me know if the suggestions improve your results. We would be able to refund the H2B antibody ab1790 if the protocol suggestions do not improve since it has been tested in rat, so I look forward to your reply.

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1-10 of 20 Q&A

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