Product nameAnti-Histone H2B (phospho S32) antibody
See all Histone H2B primary antibodies
Species reactivityReacts with: Human
Predicted to work with: Mouse
Serine 32 of Histone H2B was highlighted as being highly conserved in Cheung et al, indicating that it might be a good candidate phosphorylation site.
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 bufferPreservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS, pH 7.4
Concentration information loading...
PurityImmunogen affinity purified
Primary antibody notesSerine 32 of Histone H2B was highlighted as being highly conserved in Cheung et al, indicating that it might be a good candidate phosphorylation site.
|ELISA||Use at an assay dependent concentration. : This antibody gave a positive result in ELISA against the immunizing peptide (ab18504).|
|IHC-P||Use a concentration of 5 µg/ml. Perform heat mediated antigen retrieval before commencing with IHC staining protocol.|
|WB||1/500. Detects a band of approximately 15 kDa (predicted molecular weight: 14 kDa).|
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.
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All lanes : Anti-Histone H2B (phospho S32) antibody (ab10476) at 1/500 dilution
Lane 1 : Hela control
Lane 2 : Gamma irraditaed hela 1h
Lane 3 : Gamma irraditaed hela 2h
Lane 4 : Hela control with Human Histone H2B (phospho S32) peptide (ab18504) at 1 µg/ml
Lane 5 : Gamma irraditaed hela 1h with Human Histone H2B (phospho S32) peptide (ab18504) at 1 µg/ml
Lane 6 : Gamma irraditaed hela 2h with Human Histone H2B (phospho S32) peptide (ab18504) at 1 µg/ml
Lane 7 : Hela control with Human Histone H2B peptide (ab18507) at 1 µg/ml
Lane 8 : Gamma irraditaed hela 1h with Human Histone H2B peptide (ab18507) at 1 µg/ml
Lane 9 : Gamma irraditaed hela 2h with Human Histone H2B peptide (ab18507) at 1 µg/ml
Lysates/proteins at 25 µg per lane.
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/5000 dilution
Performed under reducing conditions.
Predicted band size: 14 kDa
Observed band size: 15 kDa why is the actual band size different from the predicted?
Exposure time: 1 minute
Western blot using ab10476 at 1/500 on HeLa lysates taken at various time points after gamma irradiation (ab13823).
ICC/IF image of ab10476 stained HeLa cells. The cells were 4% PFA fixed (10 min), permabilised in 0.1% PBS-Tween (20 min) and incubated with the antibody (ab10476, 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-rabbit 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). This antibody also gave a positive IF result in HepG2, Hek293 and MCF7 cells fixed in 4% PFA at 1ug/ml and Hela, Hek293, HepG2 and MCF7 cells fixed in 100% methanol at 1ug/ml. However, this Fast-Track antibody is not yet fully characterised. This image represents inconclusive preliminary data.
IHC image of ab10476 staining in Human Breast adenocarcinoma formalin fixed paraffin embedded tissue section, performed on a Leica 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 ab10476, 5µg/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.
ab10476 (1/200) staining Histone H2B phospho S32 in Hela cells (green). Cells were fixed in paraformaldehyde, permeabilised with 0.5% Triton X100 and counterstained with DAPI in order to highlight the nucleus (red). for further experimental details please refer to Abreview.
This product has been referenced in:
- Li J et al. Nuclear PKC-? facilitates rapid transcriptional responses in human memory CD4+ T cells through p65 and H2B phosphorylation. J Cell Sci 129:2448-61 (2016). WB, ICC/IF ; Human . Read more (PubMed: 27149922) »
- Lau AT et al. Phosphorylation of histone H2B serine 32 is linked to cell transformation. J Biol Chem : (2011). WB, ICC/IF ; Human . Read more (PubMed: 21646345) »