The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
Use 1-2µg for 106 cells.
1/1000. Predicted molecular weight: 15 kDa.
Use 1-2µg for 106 cells.
Core 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.
Belongs to the histone H3 family.
Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.
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. 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.
ChIP results obtained with ab195417 against Histone H3 (acetyl K27).
ChIP assays were performed using HeLa cells, ab195417 against Histone H3 (acetyl K27) and optimized PCR primer sets for qPCR. ChIP was performed using sheared chromatin from 1 million cells. A titration of the antibody consisting of 1, 2, 5 and 10 µg per ChIP experiment was analysed. IgG (2 µg/IP) was used as negative IP control. Quantitative PCR was performed with primers for the promoter of the active ACTB and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and TSH2B genes, used as negative controls. Figure shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis). These results are in accordance with the observation that Histone H3 (acetyl K27) acetylation is enriched at the promoters of active genes.
Immunofluorescent analysis of HeLa cells labeling Histone H3 (acetyl K27) with ab195417 at 1/500 diution. Cells were fixed with 4% formaldehyde for 10 min and blocked with PBS/TX-100 containing 5% normal goat serum and 1% BSA. The cells were immunofluorescently labelled with ab195417 in blocking solution followed by an anti-rabbit antibody conjugated to Alexa488. The middle panel shows staining of the nuclei with DAPI. A merge of the two stainings is shown on the right.
ChIP was performed on sheared chromatin from 1 million HeLaS3 cells using 2 μg of ab195417 against Histone H3 (acetyl K27). Quantitative PCR was performed with primers for the promoter of the active ACTB and EIF4A2 genes, used as positive controls, and for the coding region of the inactive MYT1 and MB genes, used as negative controls (Figure A). The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm.
The IP’d DNA was subsequently analysed on an Illumina Genome Analyzer. Library preparation, cluster generation and sequencing were performed according to the manufacturer’s instructions. The 36 bp tags were aligned to the human genome using the ELAND algorithm. Figure B shows the peak distribution along the complete X-chromosome and in two regions surrounding the ACTB and EIF4A2 positive control genes, respectively (figure C and D).
Dot Blot analysis was performed to test the cross reactivity of ab195417 against Histone H3 (acetyl K27) with peptides containing other histone modifications and the unmodified H3K27 sequence. One hundred to 0.2 pmol of the respective peptides were spotted on a membrane. The antibody was used at a dilution of 1/25000. The figure shows a high specificity of the antibody for the modification of interest.
Western blot - Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab195417)
All lanes : Anti-Histone H3 (acetyl K27) antibody - ChIP Grade (ab195417) at 1/1000 dilution
Lane 1 : HeLa whole cell extract at 40 µg Lane 2 : HeLa histone extract at 15 µg Lane 3 : Recombinant Histone H2A at 1 µg Lane 4 : Recombinant Histone H2B at 1 µg Lane 5 : Recombinant Histone H3 at 1 µg Lane 6 : Recombinant Histone H4 at 1 µg
Predicted band size: 15 kDa
The antibody was diluted in TBS-Tween containing 5% skimmed milk.