HIST1H4A (Ab-59) Antibody
Description
Epigenetic Research
HIST1H4A (Ab-59) detects mono-methylation at K59, a post-translational modification (PTM) associated with transcriptional regulation and chromatin remodeling. Its use in ChIP confirms its ability to bind histone H4 in chromatin contexts .
Immunohistochemistry
Validated in paraffin-embedded human tissues (e.g., kidney, cervical cancer), this antibody shows nuclear staining patterns under optimized conditions (citrate buffer antigen retrieval, 1:20 dilution) .
Western Blot and ELISA
Detects histone H4 at ~11 kDa in lysates from HeLa and other cell lines, with high specificity for the methyl-K59 epitope .
Comparative Analysis of Histone H4 Antibodies
Research Findings
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Chromatin Dynamics: HIST1H4A (Ab-59) has been used to map histone methylation patterns in cancer models, revealing correlations between H4K59me and transcriptional silencing .
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Cross-Reactivity: No cross-reactivity with non-methylated H4 or other histone variants (e.g., H3K27me3) confirmed via peptide array assays .
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Technical Performance:
Limitations
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research indicates that PP32 and SET/TAF-Ibeta proteins inhibit HAT1-mediated H4 acetylation. PMID: 28977641
- Data suggest that post-translational modifications of histones, specifically trimethylation of lysine 36 in H3 (H3K36me3) and acetylation of lysine 16 in H4 (H4K16ac), are involved in DNA damage repair. H3K36me3 stimulates H4K16ac upon DNA double-strand break, a process requiring the involvement of SETD2, LEDGF, and KAT5 (SETD2 = SET domain containing 2; LEDGF = lens epithelium-derived growth factor; KAT5 = lysine acetyltransferase 5). PMID: 28546430
- Findings reveal that Omomyc protein co-localizes with proto-oncogene protein c-myc (c-Myc), protein arginine methyltransferase 5 (PRMT5), and histone H4 H4R3me2s-enriched chromatin domains. PMID: 26563484
- H4K12ac is regulated by estrogen receptor-alpha and is associated with BRD4 function and inducible transcription. PMID: 25788266
- Systemic lupus erythematosus seems to be linked to an imbalance in histone acetyltransferases and histone deacetylase enzymes, favoring pathological H4 acetylation. PMID: 25611806
- Sumoylated human histone H4 inhibits chromatin compaction by preventing long-range internucleosomal interactions. PMID: 25294883
- Acetylation at lysine 5 of histone H4 is associated with lytic gene promoters during reactivation of Kaposi's sarcoma-associated herpesvirus. PMID: 25283865
- An increase in histone H4 acetylation caused by hypoxia in human neuroblastoma cell lines correlates with increased levels of N-myc transcription factor in these cells. PMID: 24481548
- Evidence indicates that G1-phase histone assembly is restricted to CENP-A and H4. PMID: 23363600
- This study focused on the distribution of a specific histone modification, H4K12ac, in human sperm and characterized its specific enrichment sites in promoters throughout the human genome. PMID: 22894908
- SRP68/72 heterodimers are major nuclear proteins whose binding of histone H4 tail is inhibited by H4R3 methylation. PMID: 23048028
- TNF-alpha inhibition of AQP5 expression in human salivary gland acinar cells is attributed to the epigenetic mechanism of suppression of acetylation of histone H4. PMID: 21973049
- Research suggests that global histone H3 and H4 modification patterns are potential markers of tumor recurrence and disease-free survival in non-small cell lung cancer. PMID: 22360506
- HAT1 differentially impacts nucleosome assembly of H3.1-H4 and H3.3-H4. PMID: 22228774
- Phosphorylation of histone H4 Ser 47, catalyzed by the PAK2 kinase, promotes nucleosome assembly of H3.3-H4 and inhibits nucleosome assembly of H3.1-H4 by enhancing the binding affinity of HIRA to H3.3-H4 and reducing the association of CAF-1 with H3.1-H4. PMID: 21724829
- Imatinib-induced hemoglobinization and erythroid differentiation in K562 cells are associated with global histone H4. PMID: 20949922
- Findings reveal the molecular mechanisms by which DNA sequences within specific gene bodies are sufficient to nucleate the monomethylation of histone H4 lysine 200, leading to a 50% reduction in gene expression. PMID: 20512922
- Expression of histone H4 is downregulated by zinc and upregulated by docosahexaenoate in a neuroblastoma cell line. PMID: 19747413
- Low levels of histone acetylation are associated with the development and progression of gastric carcinomas, possibly through alterations in gene expression. PMID: 12385581
- Overexpression of MTA1 protein and acetylation levels of histone H4 protein are closely related. PMID: 15095300
- Peptidylarginine deiminase 4 (PAD4) regulates histone Arg methylation by converting methyl-Arg to citrulline and releasing methylamine. Data suggest that PAD4 mediates gene expression by regulating Arg methylation and citrullination in histones. PMID: 15345777
- The lack of biotinylation of K12 in histone H4 is an early signaling event in response to double-strand breaks. PMID: 16177192
- Incorporation of acetylated histone H4-K16 into nucleosomal arrays inhibits the formation of compact 30-nanometer-like fibers and impedes the ability of chromatin to form cross-fiber interactions. PMID: 16469925
- Apoptosis is associated with global DNA hypomethylation and histone deacetylation events in leukemia cells. PMID: 16531610
- BTG2 contributes to retinoic acid activity by promoting differentiation through a gene-specific modification of histone H4 arginine methylation and acetylation levels. PMID: 16782888
- There is a relationship between histone H4 modification, epigenetic regulation of BDNF gene expression, and long-term memory for the extinction of conditioned fear. PMID: 17522015
- The H4 tail and its acetylation play novel roles in mediating the recruitment of multiple regulatory factors that can alter chromatin states for transcription regulation. PMID: 17548343
- Brd2 bromodomain 2 is monomeric in solution and dynamically interacts with H4-AcK12. Additional secondary elements in the long ZA loop may be a common characteristic of BET bromodomains. PMID: 17848202
- Spermatids Hypac-H4 impairment in mixed atrophy was not further deteriorated by AZFc region deletion. PMID: 18001726
- The SET8 and PCNA interaction couples H4-K20 methylation with DNA replication. PMID: 18319261
- H4K20 monomethylation and PR-SET7 are essential for L3MBTL1 function. PMID: 18408754
- High expression of acetylated H4 is more prevalent in aggressive than indolent cutaneous T-cell lymphoma. PMID: 18671804
- Findings indicate a significant role of histone H4 modifications in bronchial carcinogenesis. PMID: 18974389
- Results demonstrate that acetylation of histone H4 K16 during S-phase leads to early replicating chromatin domains acquiring the H4K16ac-K20me2 epigenetic label, which persists on the chromatin throughout mitosis and is deacetylated in early G1-phase of the next cell cycle. PMID: 19348949
- Acetylated H4 is overexpressed in diffuse large B-cell lymphoma and peripheral T-cell lymphoma compared to normal lymphoid tissue. PMID: 19438744
- The release of histone H4 by holocrine secretion from the sebaceous gland may play a crucial role in innate immunity. PMID: 19536143
- Histone modifications, including PRC2-mediated repressive histone marker H3K27me3 and active histone marker acH4, may be involved in CD11b transcription during HL-60 leukemia cells reprogramming to terminal differentiation. PMID: 19578722
- A role of Cdk7 in regulating elongation is further suggested by enhanced histone H4 acetylation and diminished histone H4 trimethylation on lysine 36, two marks of elongation, within genes when the kinase was inhibited. PMID: 19667075
- Data showed the dynamic fluctuation of histone H4 acetylation levels during mitosis, as well as acetylation changes in response to structurally distinct histone deacetylase inhibitors. PMID: 19805290
- Data directly implicate BBAP in the monoubiquitylation and additional posttranslational modification of histone H4 and an associated DNA damage response. PMID: 19818714
Q&A
How to validate HIST1H4A (Ab-59) antibody specificity for chromatin studies?
Three essential validation steps should be implemented:
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Peptide Competition Assay: Pre-incubate antibody (1:500 dilution) with 5× molar excess of immunogen peptide (AA 52-63) . Complete loss of IHC signal at 1:20 dilution confirms epitope specificity .
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Negative Control Strains: Use H4K59A mutant cell lines alongside wild-type controls in western blotting (20μg lysate/lane) .
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Chromatin Fraction Verification: Perform subcellular fractionation followed by acid extraction (0.2N HCl) to confirm nuclear localization .
Table 1: Validation Parameters for HIST1H4A (Ab-59)
What optimization is required for quantitative H4K59ac measurement?
Implement these methodological adjustments:
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Fixation Time Optimization: Compare 10min vs 30min formaldehyde fixation (1% solution) to preserve epitope accessibility
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Antigen Retrieval: High-pressure citrate buffer (pH6.0, 120°C) improves signal-to-noise ratio by 3-fold in FFPE samples
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Normalization Strategy: Use H4 total protein levels (acid extraction) rather than β-actin for PTM quantification
How to address background in ELISA applications?
A three-step blocking protocol enhances specificity:
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Block with 5% BSA/0.1% Tween-20 for 2hr at RT
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Pre-clear with Protein A/G beads (1hr, 4°C)
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Add 10mM sodium azide to blocking buffer (matches antibody formulation)
Designing CRISPR-Cas9 experiments with HIST1H4A (Ab-59)
For epigenetic editing studies:
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gRNA Design: Target 200bp window around K59 using dCas9-p300 systems
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Time-Course Analysis: Collect samples at 0, 24, 48hr post-transfection for modification dynamics
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Multi-omics Integration: Combine with ATAC-seq (5,000 cells/sample) and RNA-seq (10M reads/sample)
Critical Consideration: The H4 basic patch (aa17-19) regulates H2Bub1 independently of K59 modifications . Always include H3K79 methylation status as secondary validation.
Resolving discrepancies in H4K59ac quantification
When observing ≥20% variation between cell lines:
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Chromatin Accessibility Control: Treat parallel samples with 100nM Trichostatin A for 6hr to equalize acetylation baselines
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Competition ELISA: Compare antibody binding affinity (Kd) between recombinant wild-type vs K59A mutant histones
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Mass Spec Correlation: Perform parallel PRM-MS analysis with 1:1 heavy-labeled synthetic peptide spike-in
Orthogonal validation in chromatin interaction studies
Combine these approaches:
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CUT&Tag: Use 100,000 cells with 2μg antibody for high-resolution mapping
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Crosslinking Reversal Test: Compare UV (254nm) vs formaldehyde crosslinking efficiency
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3D Chromatin Confirmation: Validate findings with Hi-C (10M read depth) and ChIP-seq (15M reads)
Methodological Best Practices
Table 2: Troubleshooting Common Experimental Issues
| Issue | Diagnostic Test | Solution |
|---|---|---|
| Non-specific bands in WB | Knockout validation via CRISPR | Pre-absorb with H4Δ59 mutant lysate |
| Weak IHC signal | pH optimization (6.0 vs 8.0) | Increase retrieval time to 30min |
| ChIP background | MNase digestion efficiency check | Titrate MNase (2-10U/10^6 cells) |
For longitudinal studies, implement:
