Di-methyl-Histone H3.1 (K4) Recombinant Monoclonal Antibody
Description
Introduction to Di-methyl-Histone H(K4) Recombinant Monoclonal Antibody
The Di-methyl-Histone H3.1 (K4) Recombinant Monoclonal Antibody is a highly specific research tool designed to detect lysine 4 di-methylation (H3K4me2) on histone H3.1, a post-translational modification critical for chromatin structure, gene regulation, and epigenetic memory. This antibody is engineered to target the H3K4me2 epitope, enabling precise analysis of its role in transcriptional activation, developmental regulation, and disease mechanisms.
Key Features
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Target: Di-methylated lysine 4 (K4) on histone H3.1
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Applications: ChIP-seq, Western blot (WB), immunocytochemistry (ICC), immunofluorescence (IF), and intracellular flow cytometry
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Reactivity: Human, mouse, rat, and other species (varies by product)
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Type: Recombinant monoclonal antibody
Mechanism and Production
The antibody is generated via recombinant DNA technology, which ensures high specificity and reproducibility. The process involves:
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Cloning: Genes encoding the antibody’s heavy and light chains are cloned from immunized organisms or synthetic libraries.
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Expression: Host cells (e.g., mammalian or bacterial systems) produce the antibody, which is then purified using affinity chromatography.
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Validation: Rigorous testing in applications like ChIP-seq, WB, and ICC confirms target binding and minimal cross-reactivity .
Table 1: Production and Validation Overview
Applications and Optimization
The antibody is optimized for diverse experimental workflows:
Table 2: Recommended Dilutions by Application
| Application | Dilution Range | Source |
|---|---|---|
| Western Blot (WB) | 1:500–1:2000 | |
| ICC/IF | 1:50–1:500 | |
| ChIP-seq | 2–5 µg per 25–30 µg chromatin | |
| Flow Cytometry | 1:150–1:500 |
Key Notes:
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ChIP-seq: Effective for mapping H3K4me2-enriched regions in promoters and enhancers .
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ICC/IF: Reveals nuclear localization patterns, with heterogeneity observed in single cells due to dynamic chromatin states .
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WB: Detects a band at ~15–17 kDa, consistent with histone H3.1 size .
Table 3: Antibody Comparison
Advantages of Recombinant Monoclonals:
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Specificity: Minimal cross-reactivity to other histone modifications (e.g., H3K9me3) .
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Consistency: Recombinant production reduces lot-to-lot variability .
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Sensitivity: Detects low-abundance H3K4me2 marks in complex samples .
Epigenetic Regulation
H3K4me2 is linked to transcriptional activation and chromatin accessibility. Studies using ab32356 have shown:
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ChIP-seq Data: Enrichment at promoters of actively transcribed genes in HeLa cells .
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Disease Implications: Altered H3K4me2 patterns in cancer (e.g., cervical carcinoma) correlate with gene dysregulation .
Dynamic Chromatin Behavior
IF studies reveal heterogeneity in H3K4me2 staining across cells, reflecting context-dependent chromatin states .
Methodological Validation
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CUT&RUN: Demonstrated high-resolution mapping of H3K4me2 in HeLa cells .
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Flow Cytometry: Quantifies intracellular H3K4me2 levels, useful for studying epigenetic drug responses .
Critical Considerations
Product Specs
The production of the di-methyl-Histone H3.1 (K4) recombinant monoclonal antibody is a meticulously crafted process. It begins with the cloning of genes encoding the HIST1H3A antibody, encompassing both heavy and light chains. These cloned genes are then integrated into expression vectors, which are subsequently introduced into host cells through transfection. The host cells are then tasked with producing and secreting the antibody. To ensure its purity and efficacy, the antibody undergoes a rigorous purification process utilizing affinity chromatography. After purification, the antibody is subjected to comprehensive functionality testing across a range of applications, including ELISA, WB, ICC, and IF, making it a versatile tool for accurately detecting the human and mouse HIST1H3A proteins di-methylated at K4.
Di-methylation of Histone H3.1 at lysine 4 (K4) plays a pivotal role in various cellular processes, including transcriptional activation, chromatin accessibility, developmental regulation, epigenetic memory, coordinated gene regulation, and has implications in diverse disease states.
Target Background
Histone H3.1 is a core component of nucleosomes. Nucleosomes serve to wrap and compact DNA into chromatin, thereby limiting DNA accessibility to cellular machineries that rely on DNA as a template. Consequently, histones play a central role in the regulation of transcription, DNA repair, DNA replication, and chromosomal stability. DNA accessibility is regulated by a complex set of post-translational modifications of histones, often referred to as the histone code, alongside nucleosome remodeling.
- Research suggests that epigenetic regulation in cancer is mediated by the induction of E3 ubiquitin ligase NEDD4-dependent histone H3 ubiquitination. PMID: 28300060
- The detection of increased expression of H3K27me3 during a patient's clinical course can be beneficial in determining whether tumors are heterochronous. PMID: 29482987
- Studies have shown that JMJD5, a Jumonji C (JmjC) domain-containing protein, acts as a Cathepsin L-type protease that mediates histone H3 N-tail proteolytic cleavage under stress conditions that induce a DNA damage response. PMID: 28982940
- Data indicates that the Ki-67 antigen proliferative index exhibits significant limitations, and phosphohistone H3 (PHH3) emerges as an alternative proliferative marker. PMID: 29040195
- Research findings demonstrate that cytokine-induced histone 3 lysine 27 trimethylation serves as a mechanism that stabilizes gene silencing in macrophages. PMID: 27653678
- This data suggests that, in the early developing human brain, HIST1H3B constitutes the largest proportion of H3.1 transcripts among H3.1 isoforms. PMID: 27251074
- In a series of 47 diffuse midline gliomas, the histone H3-K27M mutation was found to be mutually exclusive with IDH1-R132H mutation and EGFR amplification. It rarely co-occurred with BRAF-V600E mutation and was frequently associated with p53 overexpression, ATRX loss, and monosomy 10. PMID: 26517431
- Studies demonstrate that histone chaperone HIRA co-localizes with viral genomes, binds to incoming viral, and deposits histone H3.3 onto these. PMID: 28981850
- Experiments have shown that PHF13 binds specifically to DNA and to two types of histone H3 methyl tags (lysine 4-tri-methyl or lysine 4-di-methyl), where it functions as a transcriptional co-regulator. PMID: 27223324
- Hemi-methylated CpGs DNA recognition activates UHRF1 ubiquitylation towards multiple lysines on the H3 tail adjacent to the UHRF1 histone-binding site. PMID: 27595565
- This study provides the first description of the MR imaging features of pediatric diffuse midline gliomas with histone H3 K27M mutation. PMID: 28183840
- Approximately 30% of pediatric high-grade gliomas (pedHGG), including GBM and DIPG, harbor a lysine 27 mutation (K27M) in histone 3.3 (H3.3). This mutation is correlated with poor outcome and has been shown to influence EZH2 function. PMID: 27135271
- H3F3A K27M mutation in adult cerebellar HGG is not uncommon. PMID: 28547652
- Research indicates that lysyl oxidase-like 2 (LOXL2) is a histone modifier enzyme that removes trimethylated lysine 4 (K4) in histone H3 (H3K4me3) through an amino-oxidase reaction. PMID: 27735137
- Histone H3 lysine 9 (H3K9) acetylation was most prevalent when the Dbf4 transcription level was highest, while the H3K9me3 level was greatest during and just after replication. PMID: 27341472
- The SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing. PMID: 27614073
- Data suggests that binding of the helical tail of histone 3 (H3) with PHD ('plant homeodomain') fingers of BAZ2A or BAZ2B (bromodomain adjacent to zinc finger domain 2A or 2B) requires molecular recognition of secondary structure motifs within the H3 tail and could represent an additional layer of regulation in epigenetic processes. PMID: 28341809
- Results demonstrate a novel mechanism by which Kdm4d regulates DNA replication by reducing the H3K9me3 level to facilitate the formation of the preinitiation complex. PMID: 27679476
- Histone H3 modifications caused by traffic-derived airborne particulate matter exposures in leukocytes have been observed. PMID: 27918982
- A key role of persistent histone H3 serine 10 or serine 28 phosphorylation in chemical carcinogenesis through regulating gene transcription of DNA damage response genes has been established. PMID: 27996159
- hTERT promoter mutations are frequent in medulloblastoma and are associated with older patients, prone to recurrence and located in the right cerebellar hemisphere. Conversely, histone 3 mutations do not appear to be present in medulloblastoma. PMID: 27694758
- AS1eRNA-driven DNA looping and activating histone modifications promote the expression of DHRS4-AS1 to economically control the DHRS4 gene cluster. PMID: 26864944
- Data suggests that nuclear antigen Sp100C is a multifaceted histone H3 methylation and phosphorylation sensor. PMID: 27129259
- The authors propose that histone H3 threonine 118 phosphorylation via Aurora-A alters the chromatin structure during specific phases of mitosis to promote timely condensin I and cohesin disassociation, which is essential for effective chromosome segregation. PMID: 26878753
- Hemi-methylated DNA opens a closed conformation of UHRF1 to facilitate its H3 histone recognition. PMID: 27045799
- The functional importance of H3K9me3 in hypoxia, apoptosis, and repression of APAK has been established. PMID: 25961932
- Taken together, the authors have verified that histone H3 is a true substrate for GzmA in vivo in Raji cells treated with staurosporin. PMID: 26032366
- Research shows that circulating H3 levels correlate with mortality in sepsis patients and inversely correlate with antithrombin levels and platelet counts. PMID: 26232351
- Data indicate that double mutations on the residues in the interface (L325A/D328A) decrease the histone H3 H3K4me2/3 demethylation activity of lysine (K)-specific demethylase 5B (KDM5B). PMID: 24952722
- Studies indicate that minichromosome maintenance protein 2 (MCM2) binding is not required for the incorporation of histone H3.1-H4 into chromatin but is important for the stability of H3.1-H4. PMID: 26167883
- Data suggests that histone H3 lysine methylation (H3K4me3) plays a critical mechanistic role in leukemia stem cell (LSC) maintenance. PMID: 26190263
- PIP5K1A modulates ribosomal RNA gene silencing through its interaction with histone H3 lysine 9 trimethylation and heterochromatin protein HP1-alpha. PMID: 26157143
- Data indicates that lower-resolution mass spectrometry instruments can be utilized for histone post-translational modifications (PTMs) analysis. PMID: 25325711
- Research indicates that inhibition of lysine-specific demethylase 1 activity prevented IL-1beta-induced histone H3 lysine 9 (H3K9) demethylation at the microsomal prostaglandin E synthase 1 (mPGES-1) promoter. PMID: 24886859
- The authors report that de novo CENP-A assembly and kinetochore formation on human centromeric alphoid DNA arrays are regulated by a histone H3K9 acetyl/methyl balance. PMID: 22473132
