Product nameAnti-Histone H3 (phospho T45) antibody
See all Histone H3 primary antibodies
DescriptionRabbit polyclonal to Histone H3 (phospho T45)
Tested applicationsSuitable for: WB, ICC/IF, ChIP, IHC-P, ELISAmore details
Species reactivityReacts with: Mouse, Human
Predicted to work with: Rat, Rabbit, Chicken, Cow, Pig, Drosophila melanogaster, a wide range of other species, Rainbow trout, Rice, Orangutan
- WB: HeLa Whole Cell Extract - Calyculin A Treated for 45min (20nM) IHC-P: Human normal colon FFPE tissue sections.
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
Constituents: PBS, 1% BSA
Concentration information loading...
PurityImmunogen affinity purified
ChIP Related Products
Immunizing Peptide (Blocking)
Our Abpromise guarantee covers the use of ab26127 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||Use a concentration of 1 µg/ml. Detects a band of approximately 17 kDa (predicted molecular weight: 15 kDa).|
|ICC/IF||Use at an assay dependent concentration.|
|ChIP||Use at an assay dependent concentration.|
|IHC-P||Use at an assay dependent concentration.|
|ELISA||Use at an assay dependent concentration.|
FunctionCore 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.
Sequence similaritiesBelongs to the histone H3 family.
Developmental stageExpressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
modificationsAcetylation is generally linked to gene activation. Acetylation on Lys-10 (H3K9ac) impairs methylation at Arg-9 (H3R8me2s). Acetylation on Lys-19 (H3K18ac) and Lys-24 (H3K24ac) favors methylation at Arg-18 (H3R17me).
Citrullination at Arg-9 (H3R8ci) and/or Arg-18 (H3R17ci) by PADI4 impairs methylation and represses transcription.
Asymmetric dimethylation at Arg-18 (H3R17me2a) by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 (H3R8me2s) by PRMT5 is linked to gene repression. Asymmetric dimethylation at Arg-3 (H3R2me2a) by PRMT6 is linked to gene repression and is mutually exclusive with H3 Lys-5 methylation (H3K4me2 and H3K4me3). H3R2me2a is present at the 3' of genes regardless of their transcription state and is enriched on inactive promoters, while it is absent on active promoters.
Methylation at Lys-5 (H3K4me), Lys-37 (H3K36me) and Lys-80 (H3K79me) are linked to gene activation. Methylation at Lys-5 (H3K4me) facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 (H3K79me) is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are linked to gene repression. Methylation at Lys-10 (H3K9me) is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 (H3S10ph) and acetylation of H3 and H4. Methylation at Lys-5 (H3K4me) and Lys-80 (H3K79me) require preliminary monoubiquitination of H2B at 'Lys-120'. Methylation at Lys-10 (H3K9me) and Lys-28 (H3K27me) are enriched in inactive X chromosome chromatin.
Phosphorylated at Thr-4 (H3T3ph) by GSG2/haspin during prophase and dephosphorylated during anaphase. Phosphorylation at Ser-11 (H3S10ph) by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 (H3S10ph) by RPS6KA4 and RPS6KA5 is important during interphase because it enables the transcription of genes following external stimulation, like mitogens, stress, growth factors or UV irradiation and result in the activation of genes, such as c-fos and c-jun. Phosphorylation at Ser-11 (H3S10ph), which is linked to gene activation, prevents methylation at Lys-10 (H3K9me) but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 (H3S10ph) by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 (H3S10ph) is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 (H3S28ph) by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation at Thr-7 (H3T6ph) by PRKCBB is a specific tag for epigenetic transcriptional activation that prevents demethylation of Lys-5 (H3K4me) by LSD1/KDM1A. At centromeres, specifically phosphorylated at Thr-12 (H3T11ph) from prophase to early anaphase, by DAPK3 and PKN1. Phosphorylation at Thr-12 (H3T11ph) by PKN1 is a specific tag for epigenetic transcriptional activation that promotes demethylation of Lys-10 (H3K9me) by KDM4C/JMJD2C. Phosphorylation at Tyr-42 (H3Y41ph) by JAK2 promotes exclusion of CBX5 (HP1 alpha) from chromatin.
Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination (By similarity). Ubiquitinated by the CUL4-DDB-RBX1 complex in response to ultraviolet irradiation. This may weaken the interaction between histones and DNA and facilitate DNA accessibility to repair proteins.
Cellular localizationNucleus. Chromosome.
- Information by UniProt
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ChIP analysis using ab26127 binding Histone H3 in Human RWP1 whole cell lysate. Cells were cross-linked for 5 minutes with 4% PFA. Samples were incubated with primary antibody (1/100 in 1% BSA in PBS) for 4 hours at 4°C. Protein binding was detected using real-time PCR.
Negative Control:IgG ChIP.
All lanes : Anti-Histone H3 (phospho T45) antibody (ab26127) at 1 µg
Lane 1 : HeLa treated with Calyculin A for 45min (0nM, untreated)
Lane 2 : HeLa treated with Calyculin A for 45min (20nM)
Lane 3 : HeLa treated with Calyculin A for 45min (20nM) with
Human Histone H3 (phospho T45) peptide (ab26307) at 1 µg
Lysates/proteins at 20 µg per lane.
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 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: 8 minutes
This blot was produced using a 4-12% Bis-tris gel under the MES buffer system. The gel was run at 200V for 35 minutes before being transferred onto a Nitrocellulose membrane at 30V for 70 minutes. The membrane was then blocked for an hour using 2% Bovine Serum Albumin before being incubated with abX overnight at 4°C. Antibody binding was detected using an anti-rabbit antibody conjugated to HRP, and visualised using ECL development solution ab133406.
ELISA using ab26127.
The red line indicates binding to the Histone H3 - Phospho T45 peptide (ab26307).
Binding to the following peptides was not observed:
Histone H3 (ab26308), Histone H3 peptide - phospho T3 (ab17578), Histone H3 peptide - phospho T11 (ab24444) or Histone H3 peptide - phospho T32 (ab14799)
IHC image of ab26127 staining Histone H3 in Human normal colon formalin fixed paraffin embedded tissue* sections, performed on a Leica Bond. 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 ab26127, 7µg/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. No primary antibody was used in the negative control (shown on the inset).
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.
*Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre
ab26127 staining Histone H3 (phospho T45) in HEK 293T cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 1% Triton X-100 and blocked with 3% BSA for 1 hour. Samples were incubated with primary antibody (1/50 in PBS + 3% BSA) for 1 hour. An Alexa Fluor® 488-conjugated donkey anti-rabbit IgG polyclonal (1/500) was used as the secondary antibody.
This product has been referenced in:
- Pacaud R et al. Histone H3 phosphorylation in GBM: a new rational to guide the use of kinase inhibitors in anti-GBM therapy. Theranostics 5:12-22 (2015). Read more (PubMed: 25553095) »
- Villagrasa P et al. Akt2 interacts with Snail1 in the E-cadherin promoter. Oncogene 31:4022-33 (2012). WB, ChIP ; Human . Read more (PubMed: 22158034) »