Anti-Histone H3 (phospho T11) antibody - ChIP Grade (ab5168)
Overview
- Product nameAnti-Histone H3 (phospho T11) antibody - ChIP GradeSee all Histone H3 primary antibodies ...
- DescriptionRabbit polyclonal to Histone H3 (phospho T11) - ChIP Grade
- Tested applicationsWB, ChIP, IHC-P, ICC/IF more details
- Species reactivityReacts 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 controlCalf 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
- FormLiquid
- Storage instructionsStore at +4°C short term (1-2 weeks). Aliquot and store at -20°C or -80°C. Avoid repeated freeze / thaw cycles.
- Storage bufferPreservative: 0.02% Sodium Azide
Constituents: 1% BSA, PBS. pH 7.4 -
Concentration information loading... - PurityImmunogen affinity purified
- Clonality Polyclonal
- IsotypeIgG
- Research Areas
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 | Notes |
|---|---|
| WB | WB: 1/500 - 1/1000. Detects a band of approximately 15 kDa (predicted molecular weight: 15 kDa).Can be blocked with Histone H3 peptide - phospho T11 (ab24444). |
| ChIP | ChIP: Use at an assay dependent dilution. |
| IHC-P | 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 | 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
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.
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.
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Database links
- Entrez Gene: 176359 Caenorhabditis elegans
- Entrez Gene: 326601 Cow
- Entrez Gene: 31848 Fruit fly (Drosophila melanogaster)
- Entrez Gene: 33736 Fruit fly (Drosophila melanogaster)
- Entrez Gene: 3020 Human
- Entrez Gene: 3021 Human
- Entrez Gene: 15078 Mouse
- Entrez Gene: 15081 Mouse
- Entrez Gene: 289314 Rat
- Entrez Gene: 100361558 Rat
- Entrez Gene: 100365096 Rat
- Entrez Gene: 117056 Rat
- Entrez Gene: 379062 Xenopus laevis
- Entrez Gene: 379757 Xenopus laevis
- Entrez Gene: 399418 Xenopus laevis
- Entrez Gene: 432115 Xenopus laevis
- Entrez Gene: 444503 Xenopus laevis
- Entrez Gene: 100331798 Zebrafish
- Entrez Gene: 336231 Zebrafish
- Entrez Gene: 394076 Zebrafish
- Entrez Gene: 406269 Zebrafish
- Entrez Gene: 550262 Zebrafish
- Omim: 601128 Human
- SwissProt: P59226 Arabidopsis thaliana
- SwissProt: Q10453 Caenorhabditis elegans
- SwissProt: P08898 Caenorhabditis elegans
- SwissProt: P02299 Fruit fly (Drosophila melanogaster)
- SwissProt: P84249 Fruit fly (Drosophila melanogaster)
- SwissProt: Q93081 Human
- SwissProt: Q16695 Human
- SwissProt: P84243 Human
- SwissProt: P84244 Mouse
- SwissProt: P07041 Neurospora crassa
- SwissProt: P84245 Rat
- SwissProt: Q6PI79 Xenopus laevis
- SwissProt: Q6PI20 Zebrafish
- Unigene: 2931 Fruit fly (Drosophila melanogaster)
- Unigene: 35099 Fruit fly (Drosophila melanogaster)
- Unigene: 7418 Fruit fly (Drosophila melanogaster)
- Unigene: 180877 Human
- Unigene: 533624 Human
- Unigene: 726012 Human
- Unigene: 138832 Mouse
- Unigene: 18516 Mouse
- Unigene: 315189 Mouse
- Unigene: 316825 Mouse
- Unigene: 322735 Mouse
- Unigene: 371563 Mouse
- Unigene: 442502 Mouse
- Unigene: 106155 Rat
- Unigene: 124815 Rat
- Unigene: 198918 Rat
- Unigene: 29857 Rat
- Unigene: 36404 Xenopus laevis
- Unigene: 39769 Xenopus laevis
- Unigene: 4043 Xenopus laevis
- Unigene: 55418 Xenopus laevis
- Unigene: 77438 Xenopus laevis
- Unigene: 132208 Zebrafish
- Unigene: 42924 Zebrafish
- Unigene: 75577 Zebrafish
- Unigene: 75603 Zebrafish
- Unigene: 77073 Zebrafish
see all
Target information above from: UniProt accession
P84243
The UniProt Consortium
The Universal Protein Resource (UniProt) in 2010
Nucleic Acids Res. 38:D142-D148 (2010)
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Alternative names
- H3 3 like sequence MH921 antibodyH3 3 like sequence MH921 antibodyH3 3A antibody
- H3 3A antibodyH3 a antibodyH3 b antibodyH3 c antibodyH3 d antibodyH3 f antibodyH3 h antibodyH3 histone family member E pseudogene antibodyH3 histone family member E pseudogene antibodyH3 i antibodyH3 j antibodyH3 k antibodyH3 l antibodyH33_HUMAN antibodyH3F3 antibodyH3F3 antibodyH3f3b antibodyHistone H3 3 pseudogene antibodyHistone H3 3 pseudogene antibodyHistone H3.3 antibody
see all
Anti-Histone H3 (phospho T11) antibody - ChIP Grade images
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Western blot - Histone H3 (phospho T11) antibody (ab5168)Anti-Histone H3 (phospho T11) antibody - ChIP Grade (ab5168) at 1/500 dilution + Calf thymus histone lysate
Secondary
Goat polyclonal Secondary Antibody to 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?) -
Immunocytochemistry/ Immunofluorescence - Histone H3 (phospho T11) antibody (ab5168)Darin McDonaldSKN-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.
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Western blot - Histone H3 (phospho T11) antibody (ab5168)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 withHistone H3 peptide - phospho T11 (ab24444) at 1 µg/ml
Lane 4 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg withHistone H3 peptide - phospho T11 (ab24444) at 1 µg/ml
Lane 5 : Colcemid treated HeLa Histone prep at 5 µg withHistone H3 peptide - unmodified (ab2903) at 1 µg/ml
Lane 6 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg withHistone H3 peptide - unmodified (ab2903) at 1 µg/ml
Lane 7 : Colcemid treated HeLa Histone prep at 5 µg withHistone H3 peptide - phospho S10 (ab11477) at 1 µg/ml
Lane 8 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate (ab27251) at 20 µg withHistone H3 peptide - phospho S10 (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?) -
Immunocytochemistry/ Immunofluorescence - Histone H3 (phospho T11) antibody - ChIP Grade (ab5168)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. -
Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Histone H3 (phospho T11) antibody - ChIP Grade (ab5168)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) »
