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Key Specifications Table
| Species Reactivity | Key Applications |
|---|---|
| Vrt | WB, ChIP, ChIP-seq, DB, Mplex |
| Description | |
|---|---|
| Catalogue Number | 17-677 |
| Brand Family | Upstate |
| Trade Name |
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| Description | ChIPAb+ Dimethyl-Histone H3 (Lys4) - ChIP Validated Antibody and Primer Set |
| Overview | All ChIPAb+ antibodies are individually validated for chromatin precipitation, every lot, every time. Each ChIPAb+ antibody set includes control primers (tested every lot by qPCR) to biologically validate your IP results in a locus-specific context. The qPCR protocol and primer sequences are provided, allowing researchers to validate ChIP protocols when using our antibody in their chromatin context. Each set also includes a negative control antibody to ensure specificity of the ChIP reaction. The ChIPAb+ Dimethyl-Histone H3 (Lys4) set includes the Anti-dimethyl-Histone H3 (Lys4) antibody, a negative control antibody (purified Mouse IgG), and qPCR primers which amplify a 213 bp region within the coding region of the human GAPDH gene. The dimethyl-histone H3 (Lys4) and negative control antibodies are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of dimethyl-histone H3 (Lys4)-associated chromatin. |
| Alternate Names |
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| Product Information | |
|---|---|
| Format | Purified |
| Control |
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| Presentation | Anti-dimethyl-Histone H3 (Lys4) (mouse monoclonal IgG1, clone CMA303). One vial containing 50 μg of protein G purified antibody in 50 μL PBS containing 0.05% sodium azide. Store at -20°C. Normal Mouse IgG. Two vials containing 25 μg purified Mouse IgG in 25 μL storage buffer containing 0.1% sodium azide. Store at -20°C. ChIP Primers GAPDH Coding. One vial containing 75 μL of 5 μM of each primer specific for the coding region human GAPDH. Store at -20°C. FOR: GGC TCC CAC CTT TCT CAT CC REV: GGC CAT CCA CAG TCT TCT GG |
| Quality Level | MQ100 |
| Applications | |
|---|---|
| Application | This ChIPAb+ Dimethyl-Histone H3 (Lys4) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers. |
| Key Applications |
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| Application Notes | Chromatin Immunoprecipitation: Sonicated chromatin prepared from HeLa cells (2 X 106 cell equivalents per IP) was subjected to chromatin immunoprecipitation using 2 µg of either normal mouse IgG, or Anti-dimethyl-Histone H3 (Lys4) antibody and the Magna ChIP G Kit (Cat. #17-611). Successful immunoprecipitation of dimethyl-histone H3 (Lys4)-associated DNA fragments was verified by qPCR using β-globin ChIP Primers versus GAPDH Coding primers (Please see figures). Data is presented as percent input of each IP sample relative to input chromatin for each antibody with the indicated primers. Please refer to the EZ-Magna G ChIP™ (Cat. #17-408) or EZ-ChIP™ (Cat. #17-371) protocol for experimental details. ChIP-seq Analysis: Chromatin immunoprecipitation was performed using the Magna ChIP™ HiSens kit (cat# 17-10460), 2 µg Anti-dimethyl-Histone H3 (Lys4) antibody (cat# 17-677), 20 µL Protein A/G beads, and 1e6 crosslinked HeLa cell chromatin followed by DNA purification using magnetic beads. Libraries were prepared from Input and ChIP DNA samples using standard protocols with Illumina barcoded adapters, and analyzed on Illumina HiSeq instrument. An excess of sixteen million reads from FastQ files were mapped using Bowtie (http://bowtie-bio.sourceforge.net/manual.shtml) following TagDust (http://genome.gsc.riken.jp/osc/english/dataresource/) tag removal. Peaks were identified using MACS (http://luelab.dfci.harvard.edu/MACS/), with peaks and reads visualized as a custom track in UCSC Genome Browser (http://genome.ucsc.edu) from BigWig and BED files. The highest 25% of peaks identified in the 04-790 and 17-677 datasets showed 92 and 90% overlap with peaks identified in the ENCODE H3K4me2 BROAD Histone track for HeLa S3. Western Blot Analysis: Recombinant Histone H3 (Lane 1) and HeLa acid extract (Lane 2) were resolved by electrophoresis, transferred to PVDF membrane and probed with Anti-dimethyl Histone H3 (Lys4) (2 μg/mL). Proteins were visualized using a goat anti-mouse secondary antibody conjugated to HRP (Cat. #AP124P) and a chemiluminescence detection system. Dot Blot Analysis: Absurance Histone H3 Antibody Specificity Array (Cat. No. 16-667) and Absurance Histone H2A, H2B, H4 Antibody Specificity Array (Cat. No. 16-665), which contain histone peptides with various modifications were probed with Anti-dimethyl H3 (Lys4) at 2.0ug/mL (1:500) dilution. Proteins were visualized using a Donkey anti-mouse IgG conjugated to HRP and a chemiluminescence detection system. Beadlyte® Histone Peptide Specificity Assay: 0.15 μg/ml of purified anti-dimethyl-Histone H3 (Lys4), clone CMA303 was incubated with a cocktail of microspheres conjugated to histone H3 peptides with the following modifications: 1. unmodified H3 (K4) 2. monomethyl H3 (K4) 3. dimethyl H3 (K4) 4. trimethyl H3 (K4) 5. trimethy H3 (K27) Unbound antibody was then removed by filtration. Peptide antibody complexes were incubated with a PE-conjugated anti-mouse secondary antibody. Fluorescence was read on a Luminex® 100™ instrument. Median Fluorescence intensity (MFI) is plotted. |
| Biological Information | |
|---|---|
| Immunogen | The dimethyl-histone H3 (Lys4) purified antibody is made against a synthetic peptide (dimethylated at Lys4) corresponding to amino acids 1-12 of histone H3. |
| Epitope | a.a. 1-12 |
| Clone | CMA303 |
| Host | Mouse |
| Specificity | Recognizes histone H3, Mr 17 kDa, dimethylated at lysine 4. |
| Isotype | IgG2bκ |
| Species Reactivity |
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| Species Reactivity Note | Human. The peptide sequence is identical in a wide range of animal and plant species, so broad cross-reactivity is expected. |
| Antibody Type | Monoclonal Antibody |
| Entrez Gene Number |
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| Entrez Gene Summary | Histones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. Nucleosomes consist of approximately 146 bp of DNA wrapped around a histone octamer composed of pairs of each of the four core histones (H2A, H2B, H3, and H4). The chromatin fiber is further compacted through the interaction of a linker histone, H1, with the DNA between the nucleosomes to form higher order chromatin structures. This gene is intronless and encodes a member of the histone H3 family. Transcripts from this gene lack polyA tails; instead, they contain a palindromic termination element. This gene is located separately from the other H3 genes that are in the histone gene cluster on chromosome 6p22-p21.3. |
| Modifications |
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| UniProt Number |
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| UniProt Summary | FUNCTION: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. Ref.14 Ref.18 Ref.22 SUBUNIT: The nucleosome is a histone octamer containing two molecules each of H2A, H2B, H3 and H4 assembled in one H3-H4 heterotetramer and two H2A-H2B heterodimers. The octamer wraps approximately 147 bp of DNA. Interacts with HIRA, a chaperone required for its incorporation into nucleosomes. Ref.14 SUBCELLULAR LOCATION: Nucleus. Developmental stage Expressed throughout the cell cycle independently of DNA synthesis. PTM: Acetylation is generally linked to gene activation. Acetylation on Lys-10 impairs methylation at Arg-9. Acetylation on Lys-19 and Lys-24 favors methylation at Arg-18. Citrullination at Arg-9 and/or Arg-18 by PADI4 impairs methylation and represses transcription. Asymmetric dimethylation at Arg-18 by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 by PRMT5 is linked to gene repression. Specifically enriched in modifications associated with active chromatin such as methylation at Lys-5, Lys-37 and Lys-80. Methylation at Lys-5 facilitates subsequent acetylation of H3 and H4. Methylation at Lys-80 is associated with DNA double-strand break (DSB) responses and is a specific target for TP53BP1. Methylation at Lys-10 and Lys-28, which are linked to gene repression, are underrepresented. Methylation at Lys-10 is a specific target for HP1 proteins (CBX1, CBX3 and CBX5) and prevents subsequent phosphorylation at Ser-11 and acetylation of H3 and H4. Methylation at Lys-5 and Lys-80 require preliminary monoubiquitination of H2B at Lys-120. Methylation at Lys-10 and Lys-28 are enriched in inactive X chromosome chromatin. Phosphorylated at Thr-4 by GSG2/haspin during prophase and dephosphorylated during anaphase. At centromeres, specifically phosphorylated at Thr-12 from prophase to early anaphase, probably DAPK3. Phosphorylation at Ser-11 by AURKB is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 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, which is linked to gene activation, prevents methylation at Lys-10 but facilitates acetylation of H3 and H4. Phosphorylation at Ser-11 by AURKB mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. Phosphorylation at Ser-11 is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylated at Ser-29 by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation. Phosphorylation on Ser-32 is specific to regions bordering centromeres in metaphase chromosomes. Ref.9 Ref.10 Ref.12 Ref.13 Ref.19 Ref.20 Ref.21 Ref.29 Ubiquitinated By similarity. SIMILARITY: Belongs to the histone H3 family. SEQUENCE CAUTION: The sequence CAH73371.1 differs from that shown. Reason: Erroneous gene model prediction. |
| Molecular Weight | Dimethyl-histone H3 at ~17 kDa |
| Product Usage Statements | |
|---|---|
| Quality Assurance | Chromatin Immunoprecipitation: Sonicated chromatin prepared from HeLa cells (1 X 106 cell equivalents per IP) were subjected to chromatin immunoprecipitation using 2 µg of either a normal mouse IgG, or Anti-dimethyl-Histone H3 (Lys4) antibody and the Magna ChIP G Kit (Cat. #17-611). Successful immunoprecipitation of dimethyl-histone H3 (Lys4) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH Coding (Please see figures). Please refer to the EZ-Magna G ChIP™ (Cat. #17-409) or EZ-ChIP™ (Cat. #17-371) protocol for experimental details. |
| Usage Statement |
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| Storage and Shipping Information | |
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| Storage Conditions | Stable for 1 year at -20°C from date of receipt. Aliquot upon initial thaw, avoid freeze thaw cycles. |
| Packaging Information | |
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| Material Size | 25 assays |
| Material Package | 25 assays per kit, ~2μg per chromatin immunoprecipitation |