Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)

Overview

  • Product nameAnti-Histone H2B antibody [mAbcam 52484] - ChIP Grade
    See all Histone H2B primary antibodies
  • Description
    Mouse monoclonal [mAbcam 52484] to Histone H2B - ChIP Grade
  • Tested applicationsSuitable for: IP, Flow Cyt, WB, ICC/IF, ChIP, IHC-P, IHC-Frmore details
  • Species reactivity
    Reacts with: Mouse, Rat, Chicken, Human, Drosophila melanogaster, Zebrafish
    Predicted to work with: Cow, Xenopus laevis, Caenorhabditis elegans, Orangutan
  • Immunogen

    Synthetic peptide corresponding to Human Histone H2B aa 100 to the C-terminus (C terminal) conjugated to Keyhole Limpet Haemocyanin (KLH).

  • Positive control
    • This antibody gave a positive signal in NIH3T3 and PC12 whole cell lysates and when tested against Histone H2B recombinant protein.

Properties

  • FormLiquid
  • 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.
  • Storage bufferpH: 7.40
    Preservative: 0.02% Sodium azide
    Constituent: PBS

    Please note that some batches of ab52484 may contain 0.4M arginine. Please contact Scientific Support for further information.
  • Concentration information loading...
  • PurityIgG fraction
  • ClonalityMonoclonal
  • Clone numbermAbcam 52484
  • MyelomaSp2/0
  • IsotypeIgG1
  • Research areas

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.

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

  • 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 [mAbcam 52484] - ChIP Grade images

  • ab52484 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.

    See Abreview

  • 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
    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?)
  • 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.  

  • 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).

  • 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.
  • 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
    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
  • 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
    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
  • 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.

  • 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 for Anti-Histone H2B antibody [mAbcam 52484] - ChIP Grade (ab52484)

This product has been referenced in:
  • Sawhney S  et al. Alpha-enolase is upregulated on the cell surface and responds to plasminogen activation in mice expressing a ?133p53a mimic. PLoS One 10:e0116270 (2015). WB ; Mouse . Read more (PubMed: 25643152) »
  • Hoedt E  et al. SILAC-based proteomic profiling of the human MDA-MB-231 metastatic breast cancer cell line in response to the two antitumoral lactoferrin isoforms: the secreted lactoferrin and the intracellular delta-lactoferrin. PLoS One 9:e104563 (2014). WB ; Human . Read more (PubMed: 25116916) »

See all 5 Publications for this product

Product Wall

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Human Tissue sections (Colon)
Specification Colon
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20%
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Sep 06 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Breast)
Specification Breast
Blocking step (agent) for 1 hour(s) and 0 minute(s) · Concentration: 20%
Fixative Formaldehyde
Username

Abcam user community

Verified customer

Submitted Aug 26 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 12 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 4°C
Sample Fruit fly (Drosophila melanogaster) Cell (Drosophila Embryo)
Specification Drosophila Embryo
Permeabilization No
Fixative Heat
Username

Abcam user community

Verified customer

Submitted Jul 09 2013

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Mouse Cell lysate - whole cell (Mouse lung)
Loading amount 100000 cells
Specification Mouse lung
Gel Running Conditions Reduced Denaturing
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
Username

Abcam user community

Verified customer

Submitted Nov 22 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application ChIP
Sample Human Cell lysate - whole cell (Breast cell line)
Specification Breast cell line
Type Cross-linking (X-ChIP)
Duration of cross-linking step: 0 second(s)
Detection step Real-time PCR
Username

Abcam user community

Verified customer

Submitted Nov 20 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Human Cell lysate - whole cell (Breast cell line)
Loading amount 100000 cells
Specification Breast cell line
Gel Running Conditions Non-reduced Denaturing (12.5%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5%
Username

Abcam user community

Verified customer

Submitted Nov 20 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Western blot
Sample Chicken Cell lysate - nuclear (QT6 cells)
Loading amount 7.5 µg
Specification QT6 cells
Gel Running Conditions Reduced Denaturing (12% acrylamide)
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Nov 14 2012

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Fixative Methanol
Username

Dr. Kirk McManus

Verified customer

Submitted May 09 2008

Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"