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17-10108 ChIPAb+ Dimethyl-Histone H3 (Lys27) - ChIP Validated Antibody and Primer Set, rabbit monoclonal

This ChIPAb+ Dimethyl-Histone H3 (Lys27) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.

MSDS (material safety data sheet) or SDS, CoA and CoQ, dossiers, brochures and other available documents.
Key Applications: Chromatin Immunoprecipitation (ChIP), Western Blotting, Cell Function Assay Research Category: Epigenetics & Nuclear Function Research Sub-Category: Histones
17-10108
25 assays  25 assays per set. Recommended use: ~2 μL of antibody per chromatin immunoprecipitation (dependent upon biological context).
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Species ReactivityKey Applications
VrtChIP, WB, Cell Function Assay
Description
Catalogue Number17-10108
Brand Family Upstate
Trade Name
  • ChIPAb+
  • Upstate
DescriptionChIPAb+ Dimethyl-Histone H3 (Lys27) - ChIP Validated Antibody and Primer Set, rabbit monoclonal
OverviewAll 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 (Lys27) set includes the Dimethyl-Histone H3 (Lys27) antibody, a negative control rabbit supernatant, and qPCR primers which amplify a 110 bp region of human β-globin promoter. The Dimethyl-Histone H3 (Lys27) and negative controls are supplied in a scalable "per ChIP" reaction size and can be used to functionally validate the precipitation of Dimethyl-Histone H3 (Lys27)-associated chromatin.
Alternate Names
  • H3K27me2
  • Histone H3 (di methyl K27)
  • H3 histone family, member M
  • H3 histone, family 2
  • histone 2, H3c
  • histone cluster 2, H3c
Background InformationHistones are highly conserved proteins that serve as the structural scaffold for the organization of nuclear DNA into chromatin. The four core histones, H2A, H2B, H3, and H4, assemble into an octamer (2 molecules of each). Subsequently, 146 base pairs of DNA are wrapped around the octamer, forming a nucleosome. Histones are modified post-translationally by the actions of enzymes in both the nucleus and cytoplasm. These modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. These modifications can alter local chromatin architecture, or recruit trans-acting factors that recognize specific histone modifications (the "histone code" hypothesis). The modifications occur predominantly on the N-terminal and C-terminal tails that extend beyond the nucleosome core particle. Histone H3 is methylated at Lys27 by EZH2, and and overexpression of EZH2 has been associated with both breast and prostate cancers. Methylation of H3K27 is involved in X chroosome inactivation, imprinting, circadian rhythms, and stem cell maintenance. H3K27me2 is a marker of classical heterochromatin.
Product Information
FormatCulture Supernatant
Control
  • Includes negative control rabbit supernatant and primers specific for human β-globin promoter.
PresentationAnti-Dimethyl-Histone H3 (Lys27) (rabbit monoclonal). One vial containing 50 µL of cultured supernantant in 0.05% sodium azide. Store at -20°C.

Negative Control Supernatant. One vial containing 100 µL of rabbit cultured supernatant in 0.05% sodium azide. Store at -20°C.

ChIP Primers, human β-globin. One vial containing 75 μL of 5 μM of each primer specific for the human β-globin promoter. Store at -20°C.
FOR: AGG ACA GGT ACG GCT GTC ATC
REV: TTT ATG CCC AGC CCT GGC TC
Quality LevelMQ100
Applications
ApplicationThis ChIPAb+ Dimethyl-Histone H3 (Lys27) -ChIP Validated Antibody & Primer Set conveniently includes the antibody & the specific control PCR primers.
Key Applications
  • Chromatin Immunoprecipitation (ChIP)
  • Western Blotting
  • Cell Function Assay
Application NotesChromatin Immunoprecipitation:
Representative lot data.
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using either 2 µL of Negative Control Supernatant or 2 µL of Anti-dimethyl-Histone H3 (Lys27) and the Magna ChIP™ A Kit (Cat. # 17-610).
Successful immunoprecipitation of dimethyl-Histone H3 (Lys27) associated DNA fragments was verified by qPCR using ChIP Primers, human β-globin as a positive locus, and GAPDH promoter primers as a negative locus (Please see figures). Data is presented as percent input of each IP sample relative to input chromatin for each amplicon and ChIP sample as indicated.
Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.

Western Blot Analysis:
Representative lot data.
A 1:1000-1:5000 dilution of a previous lot detected dimethyl-Histone H3 in acid extracted proteins from HeLa cells, but did not detect unmethylated recombinant Histone H3 (Catalog # 14-494).
Recombinant Histone H3 (lane 1) and HeLa cell acid precipitate (lane 2) were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-dimethyl-Histone H3 (Lys27) (1:1000 dilution).
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates dimethyl-histone H3 (~17 kDa) (Please see figures).

Peptide Inhibition Assay (PIA):
Representative lot data.
0.5-2 μM of histone H3 peptides containing dimethyl-Lys27 abolished detection of histone H3 by anti-dimethyl-Histone H3 (Lys27) (1:1000 dilution) in immunoblots of acid extracted proteins from HeLa cells.
Acid extracted proteins from HeLa cells were resolved by electrophoresis, transferred to nitrocellulose and probed with anti-dimethyl-Histone H3 (Lys27) (lane 1) or anti-dimethyl-Histone H3 (Lys27) preabsorbed with 0.5 mM of histone H3 peptides containing the following modifications:
Lane 2: dimethyl-lysine 23
Lane 3: dimethyl-lysine 27
Lane 4: dimethyl-lysine 9
A 1:1000 dilution of the primary antibody was used.
Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.
Arrow indicates dimethyl-histone H3 (~17 kDa) (Please see figures).
Biological Information
ImmunogenPeptide containing the sequence (ARme2KSA) in which me2 corresponds to dimethyl lysine at residue 27 of human histone H3.
EpitopeDimethyl Lys27
HostRabbit
SpecificityThis antibody recognizes Histone H3 dimethylated on Lys27.
IsotypeIgG
Species Reactivity
  • Vertebrates
Species Reactivity NoteBroad species cross-reactivity expected, based on sequence identity in most species.
Antibody TypeMonoclonal Antibody
Entrez Gene Number
Entrez Gene SummaryHistones are basic nuclear proteins that are responsible for the nucleosome structure of the chromosomal fiber in eukaryotes. This structure consists of approximately 146 bp of DNA wrapped around a nucleosome, an 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 found in a histone cluster on chromosome 1. This gene is one of four histone genes in the cluster that are duplicated; this record represents the telomeric copy. [provided by RefSeq]
Gene Symbol
  • H3
  • H3.2
  • H3/M
  • H3/m
  • H3/o
  • H3F2
  • H3FM
  • MGC9629
  • OTTHUMP00000014041
Modifications
  • Methylation
Purification MethodUnpurified supernatant
UniProt Number
UniProt SummaryFUNCTION: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.

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. During nucleosome assembly the chaperone ASF1A interacts with the histone H3-H4 heterodimer.

SUBCELLULAR LOCATION: Nucleus.

Developmental stage Expressed during S phase, then expression strongly decreases as cell division slows down during the process of differentiation.

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.

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.

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 by 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.

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.

SIMILARITY:Belongs to the histone H3 family.
Molecular Weight~17 kDa
Product Usage Statements
Quality AssuranceChromatin Immunoprecipitation:
Sonicated chromatin prepared from HeLa cells (1 X 10E6 cell equivalents per IP) were subjected to chromatin immunoprecipitation using either 2 µL of Negative Control Supernatant or 2 µL of Anti-dimethyl-Histone H3 (Lys27) and the Magna ChIP™ A Kit (Cat. # 17-610).
Successful immunoprecipitation of dimethyl-Histone H3 (Lys27)-associated DNA fragments was verified by qPCR using ChIP Primers, human β-globin (Please see figures).
Please refer to the EZ-Magna ChIP™ A (Cat. # 17-408) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.
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.
Storage and Shipping Information
Storage Conditions1 year at -20°C from date of shipment
Handling Recommendations: Upon receipt, and prior to removing the cap, centrifuge the vial and gently mix the solution. Aliquot into microcentrifuge tubes and store at -20°C. Avoid repeated freeze/ thaw cycles, which may damage IgG and affect product performance.
Packaging Information
Material Size25 assays
Material Package25 assays per set. Recommended use: ~2 μL of antibody per chromatin immunoprecipitation (dependent upon biological context).
Global Trade Item Number
Catalogue Number GTIN
17-10108 04053252518959