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Key Specifications Table
| Species Reactivity | Key Applications |
|---|---|
| H, M, Ma | WB, ChIP, ChIP-seq |
| Description | |
|---|---|
| Catalogue Number | 17-614 |
| Brand Family | Upstate |
| Trade Name |
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| Description | ChIPAb+ Trimethyl-Histone H3 (Lys4) - ChIP Validated Antibody and Primer Set, rabbit monoclonal |
| 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+ Trimethyl-Histone H3 (Lys4) set includes the anti-trimethyl-histone H3 (Lys4) antibody, a negative control antibody (normal rabbit serum), and qPCR primers which amplify a 166 base pair region within the promoter of the human GAPDH gene. The trimethyl-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 trimethyl-histone H3 (Lys4)-associated chromatin. |
| Alternate Names |
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| Background Information | The methylation of histones can occur on two different residues: arginine or lysine. Histone methylation can be associated with transcriptional activation or repression, depending on the methylated residue. Lysine 4 of histone H3 can be mono-, di- or trimethylated by different histone methyltransferases (HMTs) such as SET1 or ASH1. Methylation of Lys4 is often associated with transcriptional activation. The demethylase LSD1 is able to demethylate histone H3 Lys4. |
| Product Information | |
|---|---|
| Components |
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| Format | Purified |
| Control |
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| Presentation | Anti-Trimethyl-Histone H3 (Lys4) recombin-ant rabbit monoclonal IgG. One vial containing 75 μL of protein A purified rabbit IgG in storage buffer (0.1M Tris-Glycine pH 7.4, 0.15M NaCl, 0.05% NaN3, with the addition of 40% glycerol. Normal Rabbit IgG. One vial containing 75 μL of normal rabbit IgG. Control Primers. One vial containing 75 μL of 5 μM of each primer specific for human GAPDH. FOR: TAC TAG CGG TTT TAC GGG CG REV: TCG AAC AGG AGG AGC AGA GAG CGA |
| Quality Level | MQ100 |
| Applications | |
|---|---|
| Application | Trimethyl-Histone H3 (Lys4) ChIP validated antibody & primer set including the ChIP-grade antibody & the specific control PCR primers used for chromatin immunoprecipitation of H3K4Me3. |
| Key Applications |
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| Application Notes | Chromatin Immunoprecipitation: Sonicated chromatin prepared from untreated or UV treated (6 hrs, 50 joules/m2.) U2OS cells (3 X 106 cell equivalents per IP) was subjected to chromatin immunoprecipitation using 3 μL of rabbit Anti-Trimethyl Histone H3 (Lys4) and the Magna ChIP™ A (Cat. # 17-610) Kit. Immunoprecipitation of trimethyl histone H3 (Lys4) associated DNA fragments was verified by qPCR using ChIP Primers p21 flanking the human p21 promoter that contains an Sp1 binding site (Please see figures). Fold Increase is a ratio of normalized mean IP quantities extracted from standard curves derived from inputs of each chromatin sample. Trimethyl-histone (Lys4) immunoprecipitable activity associated with this promoter increases with UV treatment as observed in other studies. Please refer to the EZ-Magna A 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 (17-10460), 3 µL anti-trimethyl-Histone H3 (Lys4) antibody (cat# 17-614) or, 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 eighteen 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 17-614 and 07-473 datasets showed 99% overlap with peaks identified in the ENCODE H3K4me3 BROAD Histone track for HeLa S3. Western blot analysis and peptide inhibition: Representative blot. HeLa acid extract was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-trimethyl-histone H3 (Lys4) (1:2,000, lane 1) or preincubated with 0.4 μM Histone H3 peptide with following modifications Lane 2: monomethyl-Lysine 4, Lane 3: dimethyl Lysine 4, Lane 4: trimethyl-Lysine 4. Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system (Please see figures). |
| Biological Information | |
|---|---|
| Immunogen | The trimethyl-histone H3 (Lys4) antibody is made against a BSA-conjugated, synthetic peptide containing the sequence …RT[me3K]QT… in which me3K corresponds to trimethyl-lysine 4 of human histone H3 |
| Epitope | Trimethyl Lys4 |
| Host | Rabbit |
| Specificity | Trimethyl-Histone H3 (Lys4) |
| Isotype | IgG |
| Species Reactivity |
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| 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. Two molecules of each of the four core histones (H2A, H2B, H3, and H4) form an octamer, around which approximately 146 bp of DNA is wrapped in repeating units, called nucleosomes. The linker histone, H1, interacts with linker DNA between nucleosomes and functions in the compaction of chromatin into higher order structures. This gene contains introns and its mRNA is polyadenylated, unlike most histone genes. The protein encoded is a replication-independent member of the histone H3 family. |
| Gene Symbol |
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| UniProt Number |
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| UniProt Summary | FUNCTION: SwissProt: Q16695 # 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. SIZE: 136 amino acids; 15508 Da 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. SUBCELLULAR LOCATION: Nucleus. 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 (By similarity). & Citrullination at Arg-9 and/or Arg-18 by PADI4 impairs methylation and represses transcription (By similarity). & Asymmetric dimethylation at Arg-18 by CARM1 is linked to gene activation. Symmetric dimethylation at Arg-9 by PRMT5 is linked to gene repression (By similarity). & Methylation at Lys-5, Lys-37 and Lys-80 are linked to gene activation. 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 are linked to gene repression. 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 (By similarity). & 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. Phosphorylated at Ser-11 during the whole mitosis. Phosphorylation at Ser-11, which is linked to gene activation, prevents methylation at Lys-10 but facilitates acetylation of H3 and H4. Phosphorylated at Ser-29 by MLTK isoform 1, RPS6KA5 or AURKB during mitosis or upon ultraviolet B irradiation (By similarity). & Phosphorylation at Ser-11 is crucial for chromosome condensation and cell-cycle progression during mitosis and meiosis. In addition phosphorylation at Ser-11 is important during interphase because it enables the transcription of genes following external stimulation, like stress or growth factors. Phosphorylation at Ser-11 is also an essential regulatory mechanism for neoplastic cell transformation. Phosphorylation at Ser-11 by AURKB/Aurora-B mediates the dissociation of HP1 proteins (CBX1, CBX3 and CBX5) from heterochromatin. & Ubiquitinated (By similarity). SIMILARITY: SwissProt: Q16695 ## Belongs to the histone H3 family. |
| Molecular Weight | 17 kDa |
| Product Usage Statements | |
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| Quality Assurance | Chromatin Immunoprecipitation: Sonicated chromatin prepared from untreated HeLa cells (1 X 106 cell equivalents) was subjected to chromatin immunoprecipitation using 3 μL of either a normal rabbit IgG or 3 μL Anti-Trimethyl-Histone H3 (Lys4) Monoclonal IgG and the Magna ChIP A (Part # 17-610) Kit. Successful immunoprecipitation of trimethyl-histone H3 (Lys4) associated DNA fragments was verified by qPCR using control ChIP Primers flanking the human GAPDH promoter (Please see figures). Please refer to the EZ-Magna A ChIP™ 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 |
| Packaging Information | |
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| Material Size | 25 assays |
| Material Package | 25 assays per kit, ~3μL per chromatin immunoprecipitation |