Anti-Histone H2B 抗体 [mAbcam 64165] - ChIP Grade (ab64165)

製品の概要

製品の特性

アプリケーション

Our Abpromise guarantee covers the use of ab64165 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

アプリケーション Abreviews 特記事項
ICC/IF Use a concentration of 1 µg/ml.
WB Use a concentration of 1 - 5 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).
Flow Cyt Use 1µg for 106 cells. ab170192-Mouse monoclonal IgG2b, is suitable for use as an isotype control with this antibody.
IP Use a concentration of 5 µg/ml.
ChIP Use 5 µg for µg of chromatin.

ターゲット情報

  • 関連性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.
  • 細胞内局在Nuclear
  • 参照データベース
  • 別名
    • 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 64165] - ChIP Grade 画像

  • 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 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 ab64165.

    Secondary: Goat polyclonal to mouse IgG light chain specific (HRP) at 1/20,000 dilution.

    Band: 17kDa; Histone H2B

  • All lanes : Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165) 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/ml 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  ab64165 (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 ab64165 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 (ab64165, 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).

  • Overlay histogram showing HeLa cells stained with ab64165 (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 (ab64165, 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 IgG2b [PLPV219] (ab91366, 2µg/1x106 cells) used under the same conditions. Unlabelled sample (blue line). Acquisition of >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter. This antibody gave a positive signal in HeLa cells fixed with 4% paraformaldehyde (10 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

Anti-Histone H2B antibody [mAbcam 64165] - ChIP Grade (ab64165) 使用論文

This product has been referenced in:
  • Slopack D  et al. Forkhead BoxO transcription factors restrain exercise-induced angiogenesis. J Physiol 592:4069-82 (2014). Mouse . Read more (PubMed: 25063823) »
  • Deeg S  et al. BAG1 restores formation of functional DJ-1 L166P dimers and DJ-1 chaperone activity. J Cell Biol 188:505-13 (2010). WB ; Human . Read more (PubMed: 20156966) »

See all 2 Publications for this product

Product Wall

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 Reduced Denaturing (12.5%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 1.5%
Username

Abcam user community

Verified customer

投稿 Jun 05 2012

Abcam has not validated the combination of species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Mouse Tissue sections (Colon)
Specification Colon
Fixative Formaldehyde
Antigen retrieval step Heat mediated
Permeabilization No
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20%
Username

Abcam user community

Verified customer

投稿 Jun 05 2012

Abcam has not validated the combination of species/application used in this Abreview.
Application Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections)
Sample Human Tissue sections (Breast cell line)
Specification Breast cell line
Fixative Formaldehyde
Antigen retrieval step Heat mediated
Permeabilization No
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 20%
Username

Abcam user community

Verified customer

投稿 May 29 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: 10 minute(s) and 0 second(s)
Specification of the cross-linking agent: Formaldehyde
Detection step Real-time PCR
Username

Abcam user community

Verified customer

投稿 May 29 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (MCF10A- Breast cell line)
Specification MCF10A- Breast cell line
Fixative Methanol
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10%
Username

Abcam user community

Verified customer

投稿 May 24 2012

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

Dr. Kirk McManus

Verified customer

投稿 Dec 10 2008

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