Histones 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; and these modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. The modifications occur predominantly on the N-terminal and C-terminal tails that extend beyond the nucleosome core particle. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark, and dimethyl histone H3 Lys4 (H3K4me2) is depleted from regions with DNA methylation. Multipotential hematopoietic cells have a subset of genes that are differentially methylated (H3K4me2+/me3-). These genes are transcriptionally silent and highly enriched in lineage-specific hematopoietic genes, suggesting a role for H3K4 methylation in differentiation.
Product Information
Format
Purified
Control
HeLa Acid extract lysate
Presentation
Purified mouse monoclonal IgG1κ in PBS with 0.05% sodium azide.
Applications
Application
Anti-Dimethyl Histone H3 (Lys4) Antibody, clone CMA303 is a mouse monoclonal antibody for detection of Dimethyl Histone H3 (Lys4) also known as H3K4me2, Histone H3 (di methyl K4). Demonstrated performance in ChIP, DB, ELISA, ICC, IP, WB, Mplex.
Key Applications
Western Blotting
Chromatin Immunoprecipitation (ChIP)
Multiplexing
ELISA
Immunocytochemistry
Dot Blot
Immunoprecipitation
Application Notes
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 (Cat. # 17-611) Kit. Successful immunoprecipitation of dimethyl-histone H3 (Lys4) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH Coding.
Please refer to the EZ-Magna G ChIP™ (Cat. # 17-409) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.
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 Cat. No 05-1338, Anti-dimethyl H3 (Lys4), clone CMA303 at 2.0 µg/mL (1:500 dilution). Proteins were visualized using a Donkey anti-mouse IgG conjugated to HRP and a chemiluminescence detection system.
ChIP-seq Analysis:
Chromatin immunoprecipitation was performed using the Magna ChIP™ HiSens kit (cat# 17-10460), 2 µg of Anti-dimethyl-Histone H3 (Lys4) antibody (cat# 05-1338), 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 05-1338 datasets showed 92 and 90% overlap with peaks identified in the ENCODE H3K4me2 BROAD Histone track for HeLa S3.
Immunocytochemistry:
This antibody has been shown by an outside laboratory to be suitable for immunocytochemistry.
Immunoprecipitation:
This antibody has been shown by an outside laboratory to be suitable for immunoprecipitation.
ELISA:
This antibody has been shown by an outside laboratory to be suitable for ELISA
Multiplexing:
This antibody specifically recognizes histone H3 dimethylated on Lys4 by Luminex® assay.
Biological Information
Immunogen
Synthetic peptide corresponding to amino acids 1-12 of human Histone H3, dimethylated on Lys4, conjugated to KLH.
Epitope
Dimethylated Lys4
Clone
CMA303
Concentration
Please refer to the Certificate of Analysis for the lot-specific concentration.
Host
Mouse
Specificity
This antibody specifically recognizes Histone H3 dimethylated at Lys4. The antibody binding specificity allows for phosphorylation of Thr3 and/or modifications of Lys91.
Isotype
IgG1κ
Species Reactivity
Human Vertebrates
Species Reactivity Note
Human. Broad species cross-reactivity is expected, based on sequence homology.
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.
FUNCTION: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 STRUCTURE: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.
TISSUE SPECIFICITY:Expressed in testicular cells.
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 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: Belongs to the histone H3 family.
Product Usage Statements
Quality Assurance
Routinely evaluated by Western Blot on HeLa acid extract.
Western Blot Analysis: 2 μg/mL (1:500) dilution of this antibody detected dimethyl Histone H3 (Lys4) on 10 μg of HeLa acid extract.
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 Conditions
Stable for 1 year at 2-8°C from date of receipt. For maximum recovery of product, centrifuge the vial prior to removing the cap. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.
Histones 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; and these modifications regulate DNA transcription, repair, recombination, and replication. The most commonly studied modifications are acetylation, phosphorylation, methylation, and ubiquitination. The modifications occur predominantly on the N-terminal and C-terminal tails that extend beyond the nucleosome core particle. Dimethyl-lysine 4 histone H3 (H3K4me2) is a transcription-activating chromatin mark, and dimethyl histone H3 Lys4 (H3K4me2) is depleted from regions with DNA methylation. Multipotential hematopoietic cells have a subset of genes that are differentially methylated (H3K4me2+/me3-). These genes are transcriptionally silent and highly enriched in lineage-specific hematopoietic genes, suggesting a role for H3K4 methylation in differentiation.
Product Information
Format
Purified
Control
HeLa Acid extract lysate
Presentation
Purified mouse monoclonal IgG1κ in PBS with 0.05% sodium azide.
Applications
Application
Anti-Dimethyl Histone H3 (Lys4) Antibody, clone CMA303 is a mouse monoclonal antibody for detection of Dimethyl Histone H3 (Lys4) also known as H3K4me2, Histone H3 (di methyl K4). Demonstrated performance in ChIP, DB, ELISA, ICC, IP, WB, Mplex.
Key Applications
Western Blotting
Chromatin Immunoprecipitation (ChIP)
Multiplexing
ELISA
Immunocytochemistry
Dot Blot
Immunoprecipitation
Application Notes
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 (Cat. # 17-611) Kit. Successful immunoprecipitation of dimethyl-histone H3 (Lys4) associated DNA fragments was verified by qPCR using ChIP Primers GAPDH Coding.
Please refer to the EZ-Magna G ChIP™ (Cat. # 17-409) or EZ-ChIP™ (Cat. # 17-371) protocol for experimental details.
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 Cat. No 05-1338, Anti-dimethyl H3 (Lys4), clone CMA303 at 2.0 µg/mL (1:500 dilution). Proteins were visualized using a Donkey anti-mouse IgG conjugated to HRP and a chemiluminescence detection system.
ChIP-seq Analysis:
Chromatin immunoprecipitation was performed using the Magna ChIP™ HiSens kit (cat# 17-10460), 2 µg of Anti-dimethyl-Histone H3 (Lys4) antibody (cat# 05-1338), 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 05-1338 datasets showed 92 and 90% overlap with peaks identified in the ENCODE H3K4me2 BROAD Histone track for HeLa S3.
Immunocytochemistry:
This antibody has been shown by an outside laboratory to be suitable for immunocytochemistry.
Immunoprecipitation:
This antibody has been shown by an outside laboratory to be suitable for immunoprecipitation.
ELISA:
This antibody has been shown by an outside laboratory to be suitable for ELISA
Multiplexing:
This antibody specifically recognizes histone H3 dimethylated on Lys4 by Luminex® assay.
Biological Information
Immunogen
Synthetic peptide corresponding to amino acids 1-12 of human Histone H3, dimethylated on Lys4, conjugated to KLH.
Epitope
Dimethylated Lys4
Clone
CMA303
Concentration
Please refer to the Certificate of Analysis for the lot-specific concentration.
Host
Mouse
Specificity
This antibody specifically recognizes Histone H3 dimethylated at Lys4. The antibody binding specificity allows for phosphorylation of Thr3 and/or modifications of Lys91.
Isotype
IgG1κ
Species Reactivity
Human Vertebrates
Species Reactivity Note
Human. Broad species cross-reactivity is expected, based on sequence homology.
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.
FUNCTION: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 STRUCTURE: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.
TISSUE SPECIFICITY:Expressed in testicular cells.
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 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: Belongs to the histone H3 family.
Product Usage Statements
Quality Assurance
Routinely evaluated by Western Blot on HeLa acid extract.
Western Blot Analysis: 2 μg/mL (1:500) dilution of this antibody detected dimethyl Histone H3 (Lys4) on 10 μg of HeLa acid extract.
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 Conditions
Stable for 1 year at 2-8°C from date of receipt. For maximum recovery of product, centrifuge the vial prior to removing the cap. Avoid repeated freeze/thaw cycles, which may damage IgG and affect product performance.
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