Anti-Histone H3 (phospho S28) antibody [HTA28] (ab10543)

Overview

  • Product nameAnti-Histone H3 (phospho S28) antibody [HTA28]
    See all Histone H3 primary antibodies
  • Description
    Rat monoclonal [HTA28] to Histone H3 (phospho S28)
  • SpecificityDoes not detect the unphosphorylated epitope. It detects the phosphorylated histone molecule at the onset of mitosis (prophase, metaphase and weaker at the beginning of anaphase), but not during late anaphase.
  • Tested applicationsSuitable for: IHC-Fr, ICC/IF, WB, ICC, Flow Cytmore details
  • Species reactivity
    Reacts with: Mouse, Chicken, Hamster, Cow, Human, Drosophila melanogaster, Oikopleura - a pelagic tunicate, Marmoset (common)
  • Immunogen

    Synthetic peptide corresponding to Human Histone H3 aa 23-35 (phospho S28) conjugated to Keyhole Limpet Haemocyanin (KLH).
    Sequence:

    KAARKSA PATGGV


    Database link: P68431

  • Positive control
    • Whole extract of cultured human acute T cell leukemia Jurkat cells, treated with Nocodazole.

Properties

  • FormLiquid
  • 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: 15mM Sodium Azide
    Constituents: 1% BSA, 0.01M PBS, pH 7.4
  • Concentration information loading...
  • PurityImmunogen affinity purified
  • Purification notesPurified from culture supernatant of hybridoma cells grown in a bioreactor
  • ClonalityMonoclonal
  • Clone numberHTA28
  • IsotypeIgG2a
  • Research areas

Applications

Our Abpromise guarantee covers the use of ab10543 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
IHC-Fr Use at an assay dependent concentration.
ICC/IF Use a concentration of 2.5 µg/ml.
WB Use a concentration of 0.5 - 1 µg/ml. Detects a band of approximately 15 kDa.

We advise you to centrifuge this product vial before use.

ICC Use at an assay dependent concentration.
Flow Cyt Use at an assay dependent concentration.

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 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 i antibody
    • H3 j antibody
    • H3 k antibody
    • H3 l antibody
    • H33_HUMAN antibody
    • H3F3 antibody
    • H3f3b antibody
    • Histone H3 3 pseudogene antibody
    • Histone H3.3 antibody
    see all

Anti-Histone H3 (phospho S28) antibody [HTA28] images

  • All lanes : Anti-Histone H3 (phospho S28) antibody [HTA28] (ab10543) at 1 µg/ml

    Lane 1 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated
    Lane 2 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated with Human Histone H3 (unmodified ) peptide (ab2623) at 0.5 µg/ml
    Lane 3 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated with Human Histone H3 (phospho S28) peptide (ab5499) at 0.5 µg/ml
    Lane 4 : HeLa Histone Preparation Nuclear Lysate - Colcemid-treated with Human Histone H3 (phospho S10) peptide (ab11477) at 0.5 µg/ml

    Lysates/proteins at 2.5 µg per lane.

    Secondary
    Peroxidase Conjugated AffiniPure Rabbit Anti-Rat IgG (H+L) at 1/10000 dilution
    Developed using the ECL technique

    Performed under reducing conditions.

    Observed band size : 17 kDa (why is the actual band size different from the predicted?)


    Exposure time : 20 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 ab10543 overnight at 4°C. Antibody binding was detected using an anti-mouse antibody conjugated to HRP, and visualised using ECL development solution.

  • ab10543 staining HeLa cells by ICC/IF. The cells were fixed with paraformaldehyde and permeabilized with 0.5% Triton X100 in PBS. The cells were then stained with ab10543 at 1/1000 in PBS for 1h at 22°C. A goat anti-rat Alexa Fluro 488 (ab150157) at 1/200 was used as the secondary antibody. Nuclei are stained in red with DAPI. The antibody produces the expected mitotic-associated staining pattern and is extremely strong. MeOH fixed samples were also evaluated and produced a similar, strong staining pattern in mitotic cells.

  • ab10543 staining Histone H3 (phospho S28) in mouse embryonic brain tissue section by Immunohistochemistry (Frozen sections). Tissue samples were fixed with paraformaldehyde and blocking with 10% serum for 1 hour at 250C was performed. The sample was incubated with primary antibody (1/300)  at 40C for 16 hours. An Alexa Fluor®488-conjugated Goat polyclonal to rat IgG was used as secondary antibody at 1/500 dilution.

    See Abreview

  • ab10543 staining Histone H3 (phospho S28) in murine embryonic fibroblast cells by Immunocytochemistry/ Immunofluorescence. The cells were fixed in paraformaldehyde and permeabilised in -20°C ethanol. Samples were then incubated with primary antibody at a 1/200 dilution for 1 hour in 0.1%Saponin/ 2.5% BSA/ PBS. The secondary antibody used was a goat anti-rat IgG (H+L) conjugated to DyLight® 649 (red) used at a 1/300 dilution. Gamma tubulin (blue), Ki67 (green) using ab15580.

References for Anti-Histone H3 (phospho S28) antibody [HTA28] (ab10543)

This product has been referenced in:
  • Liu R  et al. Fstl1 is involved in the regulation of radial glial scaffold development. Mol Brain 8:53 (2015). IF . Read more (PubMed: 26382033) »
  • Knock E  et al. The methyl binding domain 3/nucleosome remodelling and deacetylase complex regulates neural cell fate determination and terminal differentiation in the cerebral cortex. Neural Dev 10:13 (2015). IHC . Read more (PubMed: 25934499) »

See all 15 Publications for this product

Product Wall

Application Immunocytochemistry/ Immunofluorescence
Blocking step BSA as blocking agent for 30 minute(s) · Concentration: 5% · Temperature: 25°C
Sample Human Cell (human)
Specification human
Permeabilization No
Fixative Parafilm
Username

Ms. Nikita Wright

Verified customer

Submitted Sep 29 2014

Application Immunocytochemistry
Sample Human Cell (HeLa)
Specification HeLa
Permeabilization Yes - 0.5% Triton X100 in PBS
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Mar 27 2014

Application Western blot
Loading amount 30 µg
Gel Running Conditions Reduced Denaturing (10%)
Sample Fruit fly (Drosophila melanogaster) Tissue lysate - whole (0-4hr embryos)
Specification 0-4hr embryos
Treatment control shRNA and wee shRNA embryos
Blocking step BSA as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 24°C
Username

Dr. Richelle Sopko

Verified customer

Submitted Mar 04 2014

Application Immunocytochemistry/ Immunofluorescence
Blocking step 1% serum, 0.1% triton, 0.1% BSA in PBS as blocking agent for 30 minute(s) · Concentration: 1% · Temperature: rt°C
Sample Human Cell (human neural progenitor cells from ips cell line)
Specification human neural progenitor cells from ips cell line
Permeabilization Yes - see blocking buffer
Fixative Paraformaldehyde
Username

Abcam user community

Verified customer

Submitted Jan 27 2014

Application Immunocytochemistry/ Immunofluorescence
Sample Human Cell (HeLa)
Specification HeLa
Permeabilization Yes - 0.5% Triton X100 in PBS
Fixative Paraformaldehyde
Username

Dr. Kirk McManus

Verified customer

Submitted Oct 03 2013

ab10543 shares between 76-92% homology with the yeast protein so both could work in yeast but this has not been tested and therefore we cannot guarantee this. If you wish to test one of this antibody, I could give you a testing discount using our AbT...

Read More
Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Neonatal Heart)
Specification Neonatal Heart
Fixative Paraformaldehyde
Permeabilization Yes - Saponin 0,1%
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted May 11 2012

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry/ Immunofluorescence
Sample Mouse Cell (mouse embryonic fibroblast (MEF))
Specification mouse embryonic fibroblast (MEF)
Fixative Paraformaldehyde
Permeabilization Yes - -20C ethanol
Username

Abcam user community

Verified customer

Submitted Aug 19 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunohistochemistry (Frozen sections)
Sample Mouse Tissue sections (Embryonic brain)
Specification Embryonic brain
Fixative Paraformaldehyde
Permeabilization Yes - 0.3% Triton x-100
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 10% · Temperature: 25°C
Username

Abcam user community

Verified customer

Submitted Jun 10 2009

Abcam guarantees this product to work in the species/application used in this Abreview.
Application Immunocytochemistry
Sample Mouse Cultured Cells (Neural stem cells)
Specification Neural stem cells
Fixative Paraformaldehyde
Permeabilization No
Blocking step Serum as blocking agent for 1 hour(s) and 0 minute(s) · Concentration: 4% · Temperature: 20°C
Username

Abcam user community

Verified customer

Submitted Dec 18 2007

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