The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use at an assay dependent concentration.
Use at an assay dependent concentration.
1/500 - 1/1000. Detects a band of approximately 17 kDa.
Variant 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.
Belongs to the histone H3 family.
Expressed throughout the cell cycle independently of DNA synthesis.
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.
Immunocytochemistry - Anti-Histone H3 (phospho T32) antibody (ab4076)This image was submitted as part of a review by Kirk McManus, University of British Columbia
Hela cells cultured on coverslips were fixed in 4% paraformaldehyde and stained with ab4076 (green) at a working dilution of 1/200. The DNA stainined with DAPI is shown in red. (100x magnification).
Western blot - Anti-Histone H3 (phospho T32) antibody (ab4076)
All lanes : Anti-Histone H3 (phospho T32) antibody (ab4076) at 1 µg/ml
Lane 1 : Colcemid treated histone calf thymus lysate Lane 2 : Untreated histone calf thymus lysate Lane 3 : Colcemid treated histone calf thymus lysate with Human Histone H3 (phospho T32) peptide (ab14799) at 1 µg/ml Lane 4 : Untreated calf thymus histone lysate with Human Histone H3 (phospho T32) peptide (ab14799) at 1 µg/ml Lane 5 : Colcemid treated calf thymus histone lysate with Human Histone H3 (unmodified ) peptide (ab2623) at 1 µg/ml Lane 6 : Untreated calf thymus histone lysate with Human Histone H3 (unmodified ) peptide (ab2623) at 1 µg/ml
Lysates/proteins at 20 µg per lane.
ab4076 recognises a band at 17 kDa, corresponding to Histone H3, in colcemid treated lysates indicating that it is a phospho-specific antibody. ab4076 is specifically blocked using the immunizing peptide (ab14799), but not the unmodified control peptide (ab2623).
This indicates that ab4079 is specifically recognising Phosphorylated T32 of Histone H3.
ab4076 specifically recognises the phosphorylated form of Histone H3
Immunocytochemistry/ Immunofluorescence - Anti-Histone H3 (phospho T32) antibody (ab4076)This image is courtesy of an Abreview submitted by Dr A Caperta
The antibody gave an excellent signal with the expected pattern in both mitotic and meiotic cells of Arabidopsis thaliana and Secale cereale (see Caperta et al. Cytogenet Genome Res 2008 122, 73-79. S. Karger AG, Basel)
Figure: Thr32 phosphorylation of histone H3 (H3T32ph) correlates with mitotic and meiotic condensation in Arabidopsis thaliana and Secale cereale. DAPI stained chromosomes are blue, H3T32ph-signals are red, and the FISH signals corresponding to the A. thaliana 180-bp centromeric repeat are green. At late prophase H3T32ph-signals coincide with entire lengths of chromosomes and the same is observed at metaphase. In diakinesis phosphorylated T32 is dispersed along bivalent arms. In the second metaphase of meiosis, the immunosignals are spread along chromosome arms. Bars=10µm.
ab4076 (4µg/ml) staining Histone H3 in human placenta using an automated system (DAKO Autostainer Plus). Using this protocol there is strong nuclear staining. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer citrate pH 6.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that, for manual staining, optimization of primary antibody concentration and incubation time is recommended. Signal amplification may be required.