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Acid-extracted histones from Hela cells were resolved by electrophoresis, transferred to nitrocellulose and probed with anti trimethyl-Histone H3 (Lys...
Chromatin Immunoprecipitation:
Sonicated Chromatin prepared from 3x106 NIH3T3 L1 cells were subjected to chromatin immunoprecipitation using 4 µg...
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ChIPAb+ Trimethyl-Histone H3 (Lys9)
| Species Reactivity | Key Applications | Host | Format | Antibody Type |
|---|---|---|---|---|
| Vrt | Chromatin Immunoprecipitation (ChIP), WB | Rabbit | Purified | Polyclonal Antibody |
Description:
ChIPAb+ Trimethyl-Histone H3 (Lys9)
Trade Name:
Upstate (Millipore)
Product 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 (Lys9) set includes the Anti-trimethyl-Histone H3 (Lys9) antibody, a negative control antibody (purified rabbit IgG), and qPCR primers which amplify a 117 bp region within the 3’ end of the human ZNF554 gene. The trimethyl-histone H3 (Lys9) 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 (Lys9) associated chromatin.
The ChIPAb+ Trimethyl-Histone H3 (Lys9) set includes the Anti-trimethyl-Histone H3 (Lys9) antibody, a negative control antibody (purified rabbit IgG), and qPCR primers which amplify a 117 bp region within the 3’ end of the human ZNF554 gene. The trimethyl-histone H3 (Lys9) 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 (Lys9) associated chromatin.
Specificity:
Recognizes histone H3, Mr 17 kDa, trimethylated at lysine 9.
Molecular Weight:
17kDa
Epitope:
Trimethyl Lys9
Immunogen:
The trimethyl-histone H3 (Lys9) purified antibody is made against BSA-conjugated, synthetic peptide containing the sequence …AR[me3K]S… in which me3K corresponds to trimethyl lysine 9 of human Histone H3.
Species Reactivity:
Vertebrates
Species Reactivity Note:
Human, mouse. The immunogen sequence is identical in a wide range of animal and plant species, so broad cross-reactivity is expected.
Application Notes:
Chromatin Immunoprecipitation:
Sonicated chromatin prepared from 2x106 HeLa cells were subjected to chromatin immunoprecipitation using 4 μg purified antibody or normal rabbit IgG and the Magna ChIP™ A kit (Cat. # 17-610).
Successful enrichment of trimethyl-Histone H3 (Lys9) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH flanking the human GAPDH promoter and primers targeting the promoter of human MyoD.
Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) kit protocols for experimental details.
Western Blot Analysis:
Recombinant Histone H3 (Lane 1) and HeLa acid extract (Lane 2) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-trimethyl-Histone H3 (Lys9), (1 μg/mL). Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system (Please see figures).
Beadlyte® Histone Peptide Specificity Assay:
0.5 μg/ml of purified anti-dimethyl-Histone H3 (Lys9) was incubated with a cocktail of microspheres conjugated to histone H3 peptides with the following modifications:
1. trimethyl-lysine 9
2. dimethyl-lysine 9
3. monomethyl-lysine 9
4. Unmodified H3
Unbound antibody was then removed by filtration. Peptide antibody complexes were incubated with a PE-conjugated anti-rabbit secondary antibody. Fluorescence was read on a Luminex® 100™ instrument. Median Fluorescence intensity (MFI) is plotted.
Sonicated chromatin prepared from 2x106 HeLa cells were subjected to chromatin immunoprecipitation using 4 μg purified antibody or normal rabbit IgG and the Magna ChIP™ A kit (Cat. # 17-610).
Successful enrichment of trimethyl-Histone H3 (Lys9) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH flanking the human GAPDH promoter and primers targeting the promoter of human MyoD.
Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) kit protocols for experimental details.
Western Blot Analysis:
Recombinant Histone H3 (Lane 1) and HeLa acid extract (Lane 2) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-trimethyl-Histone H3 (Lys9), (1 μg/mL). Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system (Please see figures).
Beadlyte® Histone Peptide Specificity Assay:
0.5 μg/ml of purified anti-dimethyl-Histone H3 (Lys9) was incubated with a cocktail of microspheres conjugated to histone H3 peptides with the following modifications:
1. trimethyl-lysine 9
2. dimethyl-lysine 9
3. monomethyl-lysine 9
4. Unmodified H3
Unbound antibody was then removed by filtration. Peptide antibody complexes were incubated with a PE-conjugated anti-rabbit secondary antibody. Fluorescence was read on a Luminex® 100™ instrument. Median Fluorescence intensity (MFI) is plotted.
Control:
Included negative control rabbit IgG antibody and control primers specific for ZNF554.
Quality Assurance:
Chromatin Immunoprecipitation:
Sonicated Chromatin prepared from 3x106 NIH3T3 L1 cells were subjected to chromatin immunoprecipitation using 4 µg of either normal rabbit IgG or Anti-trimethyl-Histone H3 (Lys9) antibody and the Magna ChIP A kit (Cat. #17-610). Successful enrichment of trimethyl-histone H3 (Lys9)-associated DNA fragments was verified by qPCR using ChIP Primers ZNF554 (Please see figures).
Please refer to the EZ-Magna A ChIP™ (Cat. #17-408) or EZ-ChIP™ (Cat. #17-371) protocol for experimental details.
Sonicated Chromatin prepared from 3x106 NIH3T3 L1 cells were subjected to chromatin immunoprecipitation using 4 µg of either normal rabbit IgG or Anti-trimethyl-Histone H3 (Lys9) antibody and the Magna ChIP A kit (Cat. #17-610). Successful enrichment of trimethyl-histone H3 (Lys9)-associated DNA fragments was verified by qPCR using ChIP Primers ZNF554 (Please see figures).
Please refer to the EZ-Magna A ChIP™ (Cat. #17-408) or EZ-ChIP™ (Cat. #17-371) protocol for experimental details.
Presentation:
Anti-trimethyl-Histone H3 (Lys9) (rabbit polyclonal IgG). One vial containing 100 μg protein A purified IgG in 100 μL of 0.02 M Phosphate buffer, pH7.4, 0.25 M NaCl, 0.05% sodium azide with 30% glycerol. Store at -20°C.
Normal Rabbit IgG. One vial containing 125 μg of normal rabbit IgG in 125 μL storage buffer containing 0.1% sodium azide. Store at -20°C.
ChIP Primers, ZNF554. One vial containing 75 μL of 5 μM of each primer specific for ZNF554. Store at -20°C.
FOR: CGG GGA AAA GCC CTA TAA AT
REV: TCC ACA TTC ACT GCA TTC GT
Normal Rabbit IgG. One vial containing 125 μg of normal rabbit IgG in 125 μL storage buffer containing 0.1% sodium azide. Store at -20°C.
ChIP Primers, ZNF554. One vial containing 75 μL of 5 μM of each primer specific for ZNF554. Store at -20°C.
FOR: CGG GGA AAA GCC CTA TAA AT
REV: TCC ACA TTC ACT GCA TTC GT
Storage Conditions:
Stable for 1 year at -20°C from date of receipt. Aliquot upon thawing, avoid freeze thaw cycles.
UniProt Number:
Entrez Gene Number:
Gene Symbol:
- H3F3A
- MGC87783
- H3.3A
- MGC87782
- H3F3
- H3.3B
- H3F3B
Alternate Names:
- H3K9me3
- Histone H3 (tri methyl K9)
Components:
- Anti-trimethyl Histone H3 (Lys9) polyclonal, 1 vial
- ZNF554 primer set, 1 vial
Usage Statement:
Unless otherwise stated in our catalog or other company documentation accompanying the product(s), our products are intended for research use only and are not to be used for any other purpose, which includes but is not limited to, unauthorized commercial uses, in vitro diagnostic uses, ex vivo or in vivo therapeutic uses or any type of consumption or application to humans or animals.
Key Applications:
- Chromatin Immunoprecipitation (ChIP)
- Western Blotting
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.
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.
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.
Brand Family:
Upstate
Product Name:
ChIPAb+ Trimethyl-Histone H3 (Lys9)
Antibody Type:
Polyclonal Antibody
Qty/Pk:
25 assays
Format:
Purified
Host:
Rabbit
Packaging:
25 assays per kit, ~4μL per chromatin immunoprecipitation.