Acetyl-HIST1H3A (K14) Antibody
Description
Overview of Acetyl-HIST1H3A (K14) Antibody
The Acetyl-HIST1H3A (K14) antibody is a specialized immunological tool designed to detect histone H3 acetylation at lysine residue 14 (K14). This modification is a key epigenetic marker associated with active chromatin states, transcriptional activation, and DNA accessibility. The antibody targets acetylated lysine 14 within the HIST1H3A protein, a replication-dependent histone variant central to nucleosome structure and chromatin organization .
Key Features
| Parameter | Details |
|---|---|
| Host Species | Rabbit (polyclonal or monoclonal) |
| Clonality | Polyclonal (e.g., ab82501, PACO03146) or Monoclonal (e.g., ab52946) |
| Immunogen | Synthesized peptide spanning acetylated K14 (human-derived sequence) |
| Reactivity | Human, Mouse, Rat (validated across multiple commercial variants) |
| Applications | Western blot (WB), Immunofluorescence (IF), Chromatin Immunoprecipitation (ChIP), IHC |
| Dilution Range | WB: 1:500–1:2000; IF: 1:200–1:1000; ChIP: Varies by protocol |
Biological Significance of H3K14 Acetylation
Histone acetylation at lysine 14 (H3K14ac) is a hallmark of transcriptionally active chromatin. This modification neutralizes the positive charge of lysine, reducing histone-DNA interactions and promoting chromatin decondensation. Key roles include:
-
Gene Activation: Facilitates access for transcriptional machinery to promoters of actively transcribed genes .
-
DNA Repair: Linked to chromatin remodeling during repair processes .
-
Cell Cycle Regulation: Associates with replication-dependent histone variants like HIST1H3A .
H3K14ac is dynamically regulated by histone acetyltransferases (HATs) and deacetylases (HDACs), making it a critical target for studying epigenetic regulation in development, disease, and therapeutic responses .
Antibody Variants and Validation
| Antibody | Clonality | Key Applications | Species Reactivity | Observed Band Size (WB) | Citations |
|---|---|---|---|---|---|
| ab82501 (Abcam) | Polyclonal | WB, IF, ICC | Human | 17 kDa, 37 kDa (reducing) | 6 |
| PACO03146 | Polyclonal | WB, IF, ELISA, ChIP | Human, Mouse, Rat | 15 kDa (predicted) | N/A |
| ab52946 (Abcam) | Monoclonal | WB, IF, IHC, ChIP | Human, Mouse, Rat | 15 kDa (predicted) | N/A |
| OASG03482 | Polyclonal | WB, IF, ELISA | Human, Mouse, Rat | 19 kDa (Uniprot) | N/A |
Critical Observations
-
Band Variability: Observed WB bands (e.g., 17 kDa and 37 kDa in ab82501) may reflect post-translational modifications or protein complex associations .
-
Specificity: Competing with acetylated K14 peptide (e.g., ab112547) abolishes signal, confirming epitope-specific binding .
-
Cross-Reactivity: No cross-reactivity with non-acetylated H3 or other lysine residues (e.g., K9, K18) reported for K14-specific antibodies .
4.1. Chromatin Immunoprecipitation (ChIP)
-
PACO03146: Validated for ChIP to map H3K14ac-enriched regions, enabling studies on active transcriptional regulatory elements .
-
ab52946: ChIP-grade monoclonal antibody suitable for high-throughput epigenetic profiling .
4.2. Western Blotting
-
ab82501: Demonstrates robust detection of H3K14ac in Jurkat cell lysates, with peptide competition confirming specificity .
-
OASG03482: Detects endogenous levels of acetylated H3K14 with optimal dilution at 1:500–1:2000 .
4.3. Immunofluorescence (IF) and IHC
-
ab52946: Compatible with automated IHC platforms (e.g., Leica BOND™ RX), validated on multi-tissue microarrays .
-
PACO03146: Effective for visualizing nuclear H3K14ac localization in fixed cells .
Considerations for Experimental Design
-
Controls: Use peptide competition (e.g., ab112547) to confirm specificity .
-
Optimization: Dilution ranges vary by application; titrate for optimal signal-to-noise ratio .
-
Cross-Reactivity: Confirm absence of reactivity with non-acetylated H3 or other acetylated lysines (e.g., K9, K18) using negative controls .
Product Specs
Constituents: 50% Glycerol, 0.01M PBS, pH 7.4
Target Background
- Research suggests a mechanism for epigenetic regulation in cancer through the induction of E3 ubiquitin ligase NEDD4-dependent histone H3 ubiquitination. PMID: 28300060
- The identification of increased expression of H3K27me3 during a patient's clinical course can be beneficial in determining if tumors are heterochronous. PMID: 29482987
- This study reports that JMJD5, a Jumonji C (JmjC) domain-containing protein, functions 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
- These findings indicate that the Ki-67 antigen proliferative index has significant limitations, and phosphohistone H3 (PHH3) is a viable alternative proliferative marker. PMID: 29040195
- The results identify cytokine-induced histone 3 lysine 27 trimethylation as a mechanism that stabilizes gene silencing in macrophages. PMID: 27653678
- This study demonstrates that in the early developing human brain, HIST1H3B comprises the largest proportion of H3.1 transcripts among H3.1 isoforms. PMID: 27251074
- This series of 47 diffuse midline gliomas revealed that histone H3-K27M mutation was mutually exclusive with IDH1-R132H mutation and EGFR amplification, rarely co-occurred with BRAF-V600E mutation, and was commonly associated with p53 overexpression, ATRX loss, and monosomy 10. Among these K27M+ diffuse midline gliomas. PMID: 26517431
- Research shows that histone chaperone HIRA co-localizes with viral genomes, binds to incoming viral, and deposits histone H3.3 onto these. PMID: 28981850
- These experiments demonstrated 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
- For the first time, this study describes 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), which is correlated with poor outcome and was shown to influence EZH2 function. PMID: 27135271
- H3F3A K27M mutation in adult cerebellar HGG is not uncommon. PMID: 28547652
- Data show 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, whereas the H3K9me3 level was greatest during and just after replication. PMID: 27341472
- SPOP-containing complex regulates SETD2 stability and H3K36me3-coupled alternative splicing. PMID: 27614073
- This research 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
- The 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. 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. 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 seem 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
- This research 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
- Functional importance of H3K9me3 in hypoxia, apoptosis, and repression of APAK. PMID: 25961932
- Taken together, the authors verified that histone H3 is a real substrate for GzmA in vivo in the Raji cells treated by staurosporin. PMID: 26032366
- This study concludes that circulating H3 levels correlate with mortality in sepsis patients and inversely correlate with antithrombin levels and platelet counts. PMID: 26232351
- Data show that double mutations on the residues in the interface (L325A/D328A) decreases the histone H3 H3K4me2/3 demethylation activity of lysine (K)-specific demethylase 5B (KDM5B). PMID: 24952722
- Research indicates 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 suggest that histone H3 lysine methylation (H3K4me3) plays a crucial 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
- This study demonstrates 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
Q&A
How do I validate the specificity of Acetyl-HIST1H3A (K14) Antibody in my experimental system?
To confirm the antibody’s specificity, employ recombinant histone controls and peptide-blocking experiments. For example:
-
Positive controls: Use recombinant H3K14ac protein (available in ELISA kits ) to confirm antibody binding.
-
Negative controls: Compare signals with unmodified H3 or variants lacking K14 (e.g., H3K14R mutants).
-
Peptide competition: Pre-incubate the antibody with acetylated or non-acetylated K14 peptides to assess binding inhibition .
-
Cross-reactivity testing: Validate against non-human species or histone variants (e.g., H3.3 ) using acid-extracted histones or nucleosomal arrays.
Table 1: Validation Strategies for K14ac Antibody
| Method | Purpose | Controls Required |
|---|---|---|
| Recombinant protein | Confirm target recognition | H3K14ac vs. unmodified H3 |
| Peptide blocking | Assess epitope specificity | Acetylated/non-acetylated peptides |
| Species reactivity | Rule out cross-reactivity | Non-human histone extracts |
What experimental applications are most suitable for this antibody?
The antibody is optimized for ELISA, immunofluorescence (IF), and immunocytochemistry (ICC) . For ChIP, cross-reactivity with DNA-bound epitopes may require optimization.
How do I optimize antibody concentration for my assay?
Titrate the antibody using positive/negative controls:
-
ELISA: Start at 1:1,000 dilution, adjust based on signal-to-noise ratio .
-
ICC/IF: Test 1:200 to 1:500 dilutions, optimize for minimal background.
-
Western blot: Use 1:1,000 to 1:2,000 dilutions with recombinant H3K14ac as a reference.
Why does H3K4me3 loss confer resistance to TSA-induced H3K14 acetylation?
H3K4me3 directs Sgf29-dependent acetylation via its tandem tudor domain (TTD), which recognizes H3K4me3 . In Dictyostelium, disrupting H3K4me3 or Sgf29 delays H3K14ac accumulation and reduces TSA sensitivity. This crosstalk suggests H3K4me3 acts as a landing pad for acetyltransferases like SAGA.
Table 2: Key Findings on H3K4me3-K14ac Crosstalk
How do I resolve discrepancies between ELISA and ChIP data for H3K14ac?
Orthogonal validation and sample normalization are critical:
-
ELISA: Quantifies total histone acetylation, unaffected by chromatin accessibility.
-
ChIP: Detects site-specific acetylation but may miss regions with low nucleosome stability.
-
Normalization: Compare ELISA results to total histone H3 levels (e.g., via Bradford assay) and ChIP input controls.
-
Cross-verification: Use recombinant histone standards (e.g., Active Motif H3K14ac ) to calibrate both methods.
What experimental designs can elucidate the role of H3K14ac in chromatin structure?
Investigate nucleosome stability and chromatin compaction:
-
In vitro assays: Reconstitute nucleosomes with H3K14ac and measure compaction via AUC or cryo-EM .
-
Live-cell imaging: Track chromatin dynamics post-TSA treatment using H3K14ac IF.
-
Genome-wide profiling: Perform cut&RUN or ChIP-seq to map H3K14ac co-occurrence with other modifications (e.g., H3K4me3, H3K27ac).
How does H3K14 acetylation interact with other histone modifications?
Acetylation at K14 antagonizes charge-based nucleosome-DNA interactions, potentially synergizing with H3K4me3 (activating) or H3K27me3 (repressive) to modulate gene activity. For example:
-
H3K4me3/K14ac: Marks active enhancers/promoters.
-
H3K27me3/K14ac: May define poised regulatory regions.
