The antibody specifically recognizes the acetylated lysine 14 (K14ac) modification on histone H3, a core component of nucleosomes. This PTM is associated with open chromatin states, facilitating DNA accessibility for transcription machinery .
Developed for high specificity and sensitivity, this polyclonal antibody exhibits robust performance across multiple experimental platforms:
Validated in HeLa cells, showing dose-dependent enrichment at active promoters (e.g., ACTB, GAPDH) and no binding to heterochromatic regions (e.g., Sat2 satellite repeats) .
Recovery rates: 0.5–5 μg antibody per ChIP yielded 0.8–2.5% input DNA for active promoters .
Localizes acetylated H3K14 to euchromatic regions in interphase nuclei, correlating with transcriptional activity .
Synergy with Phosphorylation: H3K14ac cooperates with H3S10 phosphorylation (H3S10ph) to recruit 14-3-3 proteins, displacing repressive HP1γ complexes and activating transcription (e.g., HDAC1 gene) .
Disease Relevance: Aberrant H3K14ac levels are implicated in cancer and neurodegenerative disorders, making this antibody critical for epigenetic research .
Specificity: No cross-reactivity with H3K9ac, H3K27ac, or unmodified H3K14 in dot blot assays .
Batch Consistency: Recombinant formats ensure uniform performance across lots .
Automated Platforms: Validated on the Leica BOND™ RX system for high-throughput IHC .
HIST1H3A (also known as H3.1) is one of the main histone H3 variants found in mammals. As a core component of nucleosomes, it plays central roles in chromatin structure, gene regulation, DNA repair, DNA replication, and chromosomal stability . The protein has an observed molecular weight of approximately 15-17 kDa, though it typically appears around 17 kDa on Western blots .
Histone H3.1 contains numerous sites for post-translational modifications (PTMs), including acetylation, methylation, and phosphorylation, which collectively constitute part of the "histone code" that regulates chromatin dynamics and transcriptional states .
Histone H3.1 and H3.3 demonstrate distinct genomic localization patterns associated with their specific functions:
Feature | Histone H3.1 | Histone H3.3 |
---|---|---|
Genomic localization | Coincides with repressive marks (H3K9me3, H3K27me3, DNA methylation) | Colocalizes with activation marks (H3K4me3, H2BK120ub1, RNA pol II) |
Deposition timing | DNA synthesis-dependent (during S-phase) | DNA synthesis-independent (throughout cell cycle) |
Function | Canonical nucleosome formation during replication | Replacement histone outside S-phase, during transcription |
Aberrant localization of these variants is associated with certain cancers . This distinction is crucial when selecting antibodies for experiments examining specific chromatin states or dynamic processes.
Acetylation of histone H3 at lysine 14 (H3K14ac) is primarily associated with transcriptional activation . Research has shown that:
H3K14ac works synergistically with other modifications, particularly H3S10 phosphorylation, to create binding sites for effector proteins like 14-3-3 that mediate gene activation .
H3K14ac can co-exist with H3K9 methylation in vivo, suggesting it can help overcome repressive methylation marks .
H3K14ac is resistant to deacetylation by certain histone deacetylase complexes (like the CoREST complex) when present in nucleosomal contexts, providing a mechanism for maintaining active chromatin states .
These findings highlight H3K14ac as a critical modification in establishing and maintaining transcriptionally active chromatin domains.
The HIST1H3A (Ab-14) antibody has been validated for multiple applications with specific recommended dilutions:
Application | Recommended Dilution | Sample Type |
---|---|---|
Western Blotting (WB) | 1:500-1:2000 | Cell lysates, tissue extracts |
Immunofluorescence (IF)/ICC | 1:50-1:500 | Fixed cells |
Chromatin Immunoprecipitation (ChIP) | 1:50 | Crosslinked chromatin (10 μl antibody per 10 μg chromatin) |
Flow Cytometry (FC) | 0.80 μg per 10^6 cells | Cell suspensions |
ELISA | Varies by assay design | Purified histones, peptides |
For optimal results in ChIP experiments, use 10 μl of antibody with approximately 10 μg of chromatin (roughly 4 × 10^6 cells) . Always titrate the antibody for your specific experimental system to determine optimal concentrations.
For successful ChIP experiments targeting H3K14ac:
Cell preparation: Harvest approximately 4 × 10^6 cells per immunoprecipitation reaction.
Crosslinking: Fix cells with 1% formaldehyde for 10 minutes at room temperature to preserve protein-DNA interactions.
Chromatin preparation:
Lyse cells and isolate nuclei
Sonicate chromatin to fragments of 200-500 bp
Check fragmentation quality on an agarose gel
Immunoprecipitation:
Washing and elution:
Wash beads with increasing stringency buffers
Elute protein-DNA complexes
Reverse crosslinks and purify DNA for downstream analysis
Include appropriate controls such as IgG negative control and a positive control (e.g., H3 total antibody) to validate specificity and efficiency.
To analyze combinatorial histone modifications:
Histone H3 PTM Multiplex Assay: This approach works as a solution-based sandwich ELISA to detect multiple histone modifications simultaneously:
Sample preparation: Dilute histone samples in assay buffer with deacetylase, protease, and phosphatase inhibitors to preserve modification states.
Normalization: Include histone H3 total antibody-conjugated beads for normalization across samples .
This approach allows simultaneous detection of modifications like H3K14ac alongside other marks such as H3K9ac or H3S10ph, enabling analysis of how these modifications interact and co-occur.
Inconsistent H3K14ac detection can result from several factors:
Dynamic modification status: H3K14ac levels change rapidly with cellular conditions. Anisomycin treatment combined with HDAC inhibitors like TSA significantly increases H3K14ac levels, particularly in combination with S10 phosphorylation .
Epitope masking: The presence of neighboring modifications can affect antibody access. For example, phosphorylation at S10 can alter the recognition of K14ac by some antibodies.
Cell fixation issues: Improper fixation can lead to loss of histone modifications or reduced epitope accessibility. For immunofluorescence, different fixation methods may yield varying results:
Deacetylase activity: Endogenous HDAC activity can reduce acetylation levels during sample preparation. Always include deacetylase inhibitors in buffers when working with acetylated histones.
Validating antibody specificity is critical for reliable results:
Peptide competition assay: Pre-incubate the antibody with H3K14ac peptide before application to samples. This should abolish specific binding, as demonstrated with the Jurkat cell lysate Western blot .
Knockout/knockdown validation: Use CRISPR/Cas9-generated H3K14 mutants or HAT inhibitors to reduce K14ac levels.
Multiple antibody comparison: Use different antibodies targeting the same modification from various vendors to confirm consistent patterns.
Mass spectrometry correlation: Compare antibody-based detection with mass spectrometry quantification of histone modifications.
Modified vs. unmodified controls: Include both acetylated and non-acetylated histone H3 controls to demonstrate specificity.
H3K14 acetylation participates in complex cross-talk with other histone modifications:
Synergy with S10 phosphorylation: H3S10 phosphorylation combined with H3K14 acetylation (phosphoacetylation) creates a high-affinity binding site for 14-3-3 proteins. This binding is necessary for the transcriptional activation of specific genes, including HDAC1 .
Antagonism with K9 methylation: While H3K9 methylation is typically associated with gene repression, H3K14ac can co-exist with K9 methylation, creating a "phosphomethylation" state when S10 is also phosphorylated. This suggests that acetylation and phosphorylation can synergize to overcome repressive methylation marks .
Differential susceptibility to deacetylases: The CoREST complex (containing HDAC1 and LSD1) shows marked preference for H3 acetyl-K9 versus acetyl-K14 in nucleosome substrates. This selective resistance to deacetylation may help maintain active chromatin states .
These interactions constitute part of the "binary switching model" where one modification can influence the recognition or establishment of another, creating a dynamic code for transcriptional regulation.
14-3-3 proteins are crucial mediators of histone phosphoacetylation signals:
Binding specificity: 14-3-3 proteins (particularly 14-3-3ζ and 14-3-3ε) bind to histone H3 in a modification-dependent manner. S10 phosphorylation is necessary for interaction, but additional K14 acetylation significantly increases binding affinity .
Recruitment to target genes: ChIP experiments reveal that 14-3-3 proteins are recruited to genes like HDAC1 in an H3S10ph-dependent manner, with recruitment enhanced by additional H3 acetylation .
Displacement of repressive factors: 14-3-3 recruitment correlates with dissociation of the repressive binding module HP1γ from chromatin, suggesting a mechanism for switching from repressive to active states .
Functional requirement: siRNA-mediated depletion of 14-3-3ζ abolishes transcriptional activation of HDAC1, demonstrating that 14-3-3 proteins are essential mediators of the phosphoacetylation signal .
This represents a mechanistic link between specific histone modifications and the recruitment of effector proteins that drive changes in chromatin structure and gene expression.
Innovative approaches using hydroxamic acid (Hd) warheads have revealed insights into HDAC selectivity:
Hydroxamic acid analogs: By incorporating the Lys analog AsuHd (2-aminosuberic acid ω-hydroxamate) into semisynthetic histone H3 at positions 9 and 14, researchers created nucleosomes containing H3K9Hd or H3K14Hd .
Inhibition assays: Both H3K9Hd and H3K14Hd nucleosomes inhibit LHC (LSD1-HDAC1-CoREST complex) deacetylase activity with sub-micromolar potencies, while unmodified or acetylated nucleosomes show little effect under the same conditions .
Competitive inhibition: H3K14Hd acts as a competitive inhibitor versus peptide substrates for the LHC reaction, providing mechanistic insights into enzyme-substrate interactions .
This approach reveals that the diminished activity of histone deacetylase complexes toward H3K14ac in nucleosomes is not merely due to steric accessibility, suggesting intrinsic recognition properties of the enzyme complex for different modification sites.
H3K14 acetylation plays important roles in cellular signaling cascades:
MAP kinase pathways: The nucleosomal response links H3S10 phosphorylation (often coupled with K14 acetylation) to activation of immediate early genes like c-fos and c-jun in response to growth factors, phorbol esters, phosphatase inhibitors, and protein synthesis inhibitors .
Signal-specific kinase involvement: The nucleosomal response is mediated by either the extracellular signal-regulated kinase (ERK) or p38 MAP kinase cascades, depending on the stimulus .
Stimulus-induced specificity: Unlike global mitotic phosphorylation of histone H3, stimulus-induced H3 phosphoacetylation targets only a small fraction of nucleosomes, specifically at genes responding to the stimulus .
Kinetics correlation: The kinetics of histone H3 phosphoacetylation closely parallel the expression profiles of induced genes, suggesting a direct mechanistic link .
Understanding these pathways provides insights into how extracellular signals are transmitted to chromatin to induce specific gene expression programs.
Recent research has uncovered unexpected enzymatic properties of histone H3:
Copper binding and reduction: The histone H3-H4 tetramer functions as a copper reductase enzyme, containing a copper-binding site involving H3H113 .
Functional significance: Mutation of H3H113 to alanine is lethal in Saccharomyces cerevisiae, while H3H113N or H3H113Y mutations result in viable but slow-growing yeast strains .
Loss of function correlation: The H3H113N and H3H113Y mutations result in loss of Cu2+ binding in vitro, coinciding with their loss-of-function phenotypes in vivo .
This unexpected enzymatic activity of histone H3 suggests additional roles beyond its structural function in nucleosomes, potentially linking chromatin structure to cellular metabolism and redox homeostasis.
For optimal preservation and detection of H3K14ac:
Cell harvesting:
For adherent cells: Scrape cells in PBS containing phosphatase inhibitors
For suspension cells: Collect by centrifugation at 4°C
Histone extraction:
Acid extraction method: Lyse cells in Triton Extraction Buffer (PBS containing 0.5% Triton X-100, 2mM PMSF, 0.02% NaN₃)
Incubate on ice for 10 minutes
Centrifuge at 6,500 x g for 10 minutes
Resuspend pellet in 0.2N HCl
Extract overnight at 4°C
Centrifuge and neutralize supernatant with 1/10 volume of 2M NaOH
Sample storage:
Store extracted histones at -80°C with protease inhibitors, HDAC inhibitors (e.g., sodium butyrate), and phosphatase inhibitors
For long-term storage, add 50% glycerol
Modification enhancement:
For quantitative analysis of histone modifications:
Western blot quantification:
Use total H3 antibody for normalization
Include a standard curve of recombinant histones
Analyze bands using densitometry software
Present results as ratio of H3K14ac to total H3
ChIP-seq analysis:
Normalize to input control and IgG background
Use spike-in controls (e.g., Drosophila chromatin) for cross-sample normalization
Apply appropriate statistical methods for peak calling
Compare enrichment at specific genomic features (promoters, enhancers, etc.)
Multiplex bead-based assays:
Immunofluorescence quantification:
Use DAPI staining to define nuclear regions
Measure mean fluorescence intensity within nuclear regions
Normalize to total H3 staining in parallel samples
Apply appropriate statistical tests for population analysis
These quantitative approaches enable robust comparative analysis of histone modification levels across different experimental conditions.