Anti-Histone H3 (phospho T11) antibody - ChIP Grade (ab5168)

Overview

  • Product nameAnti-Histone H3 (phospho T11) antibody - ChIP GradeSee all Histone H3 primary antibodies ...
  • Description
    Rabbit polyclonal to Histone H3 (phospho T11) - ChIP Grade
  • Tested applicationsWB, ChIP, IHC-P, ICC/IFmore details
  • Species reactivity
    Reacts with: Mouse, Cow, Human
    Predicted to work with: Rat, Saccharomyces cerevisiae, Xenopus laevis, Arabidopsis thaliana, Caenorhabditis elegans, Fruit fly (Drosophila melanogaster), Schizosaccharomyces pombe, Zebrafish, Neurospora crassa
  • Immunogen

    Synthetic peptide conjugated to KLH derived from within residues 1 - 100 of Human Histone H3, phosphorylated at T11.

  • Positive control
    • Calf thymus histone lysate; Colcemid treated HeLa Histone prep; HeLa (Human epithelial carcinoma cell line) Nuclear Lysate IHC-P: human normal testis FFPE tissue sections

Properties

Applications

Our Abpromise guarantee covers the use of ab5168 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 1/500 - 1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).Can be blocked with Human Histone H3 (phospho T11) peptide (ab24444).
ChIP Use at an assay dependent dilution.
IHC-P Use a concentration of 1 µg/ml. Perform heat mediated antigen retrieval with citrate buffer pH 6 before commencing with IHC staining protocol.
ICC/IF Use a concentration of 1 µg/ml.

Target

  • FunctionVariant histone H3 which replaces conventional H3 in a wide range of nucleosomes in active genes. Constitutes the predominant form of histone H3 in non-dividing cells and is incorporated into chromatin independently of DNA synthesis. Deposited at sites of nucleosomal displacement throughout transcribed genes, suggesting that it represents an epigenetic imprint of transcriptionally active chromatin. 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 throughout the cell cycle independently of DNA synthesis.
  • Post-translational
    modifications
    Acetylation 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.
    Specifically enriched in modifications associated with active chromatin such as methylation at Lys-5 (H3K4me), Lys-37 and Lys-80. 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), which are linked to gene repression, are underrepresented. 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. Phosphorylation on Ser-32 (H3S31ph) is specific to regions bordering centromeres in metaphase chromosomes.
    Ubiquitinated. Monoubiquitinated by RAG1 in lymphoid cells, monoubiquitination is required for V(D)J recombination.
  • Cellular localizationNucleus. Chromosome.
  • Information by UniProt
  • Database links
    see all
  • Alternative names
    • H3 3 like sequence MH921 antibody
    • H3 3 like sequence MH921 antibody
    • H3 3A antibody
    • H3 3A antibody
    • H3 a antibody
    • H3 b antibody
    • H3 c antibody
    • H3 d antibody
    • H3 f antibody
    • H3 h antibody
    • H3 histone family member E pseudogene antibody
    • H3 histone family member E pseudogene antibody
    • H3 i antibody
    • H3 j antibody
    • H3 k antibody
    • H3 l antibody
    • H33_HUMAN antibody
    • H3F3 antibody
    • H3F3 antibody
    • H3f3b antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3.3 antibody
    see all

Anti-Histone H3 (phospho T11) antibody - ChIP Grade images

  • Anti-Histone H3 (phospho T11) antibody - ChIP Grade (ab5168) at 1/500 dilution + Calf thymus histone lysate

    Secondary
    Goat Anti-Rabbit IgG H&L (HRP) (ab6721) at 1/2000 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?)
  • SKN-SH cells were fixed in 4% paraformaldehyde for 10 mins, permeabilized in PBS-0.5% Triton X-100 for 5 mins and incubated for 30 minutes with ab5168 (1/100). The slides were rinsed once in PBS-Triton (0.1%), twice in PBS then incubated with the secondary antibody for 30 mins. The DNA is stained with DAPI (blue). Clear nuclear staining with ab5168 can be seen (green). 100x magnification.

  • All lanes : Anti-Histone H3 (phospho T11) antibody - ChIP Grade (ab5168) at 1 µg/ml

    Lane 1 : Colcemid treated HeLa Histone prep at 5 µg
    Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg
    Lane 3 : Colcemid treated HeLa Histone prep at 5 µg with Human Histone H3 (phospho T11) peptide (ab24444) at 1 µg/ml
    Lane 4 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg with Human Histone H3 (phospho T11) peptide (ab24444) at 1 µg/ml
    Lane 5 : Colcemid treated HeLa Histone prep at 5 µg with Human Histone H3 (unmodified ) peptide (ab2903) at 1 µg/ml
    Lane 6 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg with Human Histone H3 (unmodified ) peptide (ab2903) at 1 µg/ml
    Lane 7 : Colcemid treated HeLa Histone prep at 5 µg with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml
    Lane 8 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg with Human Histone H3 (phospho S10) peptide (ab11477) at 1 µg/ml

    Secondary
    Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed at 1/10000 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?)
  • ICC/IF image of ab5168 stained Hek293 cells. The cells were 4% PFA fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab5168, 1µg/ml) overnight at +4°C. 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) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in 4% PFA fixed (10 min) HeLa and HepG2 cells at 1µg/ml, and in 100% methanol fixed (5 min) HeLa, Hek293, HepG2 and MCF7 cells at 1µg/ml.
  • IHC image of Histone H3 (phospho T11) staining in human normal testis 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 ab5168, 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.

    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.

References for Anti-Histone H3 (phospho T11) antibody - ChIP Grade (ab5168)

This product has been referenced in:
  • Dastidar EG  et al. Comprehensive histone phosphorylation analysis and identification of Pf14-3-3 protein as a histone H3 phosphorylation reader in malaria parasites. PLoS One 8:e53179 (2013). WB . Read more (PubMed: 23308157) »
  • Filippakopoulos P  et al. Histone recognition and large-scale structural analysis of the human bromodomain family. Cell 149:214-31 (2012). WB . Read more (PubMed: 22464331) »

See all 8 Publications for this product

Product Wall

Application Flow Cytometry
Fixation Paraformaldehyde
Permeabilization Yes - eBioscience Permeabilization Buffer
Sample Human Cell (Differentiated Haematopoietic Stem Cells)
Specification Differentiated Haematopoietic Stem Cells
Gating Strategy Isotype negative control (white)
Preparation Cell harvesting/tissue preparation method: Typsin-EDTA Cell Dissociation
Sample buffer: PBS and 10% FBS
Username

Abcam user community

Verified customer

Submitted Feb 27 2014

Application Western blot
Loading amount 20 µg
Gel Running Conditions Reduced Denaturing
Sample Human Tissue lysate - whole (Patient Dermal Fibroblasts)
Specification Patient Dermal Fibroblasts
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
Username

Abcam user community

Verified customer

Submitted Feb 05 2014

Application Western blot
Sample Mouse Cell lysate - whole cell (Mouse ES Cells)
Loading amount 50 µg
Specification Mouse ES Cells
Gel Running Conditions Reduced Denaturing (15% SDS PAGE)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 25°C
Username

Mr. Utkarsh Kapoor

Verified customer

Submitted Dec 26 2012

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...

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Thank you for contacting us.

I am sorry to hear that the antibody is not working as expected. As we discussed, please let me know which antibody you would like to receive as free of charge replacement, and I'd be happy to arrange the shipmen...

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Application Western blot
Sample Mouse Cell lysate - whole cell (fibroblast)
Loading amount 22 µg
Specification fibroblast
Gel Running Conditions Reduced Denaturing (15%)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 22°C
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Abcam user community

Verified customer

Submitted Aug 03 2010

Application Western blot
Sample Human Cell lysate - whole cell (HT29 tumour cells)
Loading amount 50 µg
Specification HT29 tumour cells
Treatment 50nM SN38 for 6h +/- 50nM staurosporine
Gel Running Conditions Reduced Denaturing (16% Tris Glycine)
Blocking step Milk as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 5% · Temperature: 23°C
Username

Abcam user community

Verified customer

Submitted May 28 2009

Thank you for your enquiry. I would recommend that your customer performs a histone extraction prior to performing western blotting. This would serve as an excellent positive control for the purpose of detection. I would direct them to the followin...

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Thank you for your enquiry. Whilst we do not have a recommended positive control for this antibody. I would like to recommend that a calf thymus histone lysate is used. This is shown in the western blot on the datasheet. Alternatively histones can b...

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