HIST1H4A (Ab-12) Antibody

What is HIST1H4A (Ab-12) Antibody and what epitope does it recognize?

HIST1H4A (Ab-12) Antibody is a polyclonal antibody raised in rabbits that specifically recognizes the acetylated form of lysine 12 (acLys12) on histone H4 . This antibody binds to a peptide sequence surrounding the acetylated lysine 12 site derived from human histone H4 . Histone H4 is a core histone protein that forms part of the nucleosome structure, and acetylation at lysine 12 represents an important post-translational modification with significant implications for chromatin regulation . The antibody has been validated for reactivity with human samples, and depending on the specific clone, may also cross-react with mouse and rat samples due to the high conservation of histone proteins across species .

What applications can HIST1H4A (Ab-12) Antibody be used for in research?

The HIST1H4A (Ab-12) antibody has been validated for multiple applications, making it versatile for various experimental approaches in epigenetic research. These applications include Enzyme-Linked Immunosorbent Assay (ELISA), Western Blotting (WB), Immunohistochemistry (IHC), Immunofluorescence (IF), Immunoprecipitation (IP), and Chromatin Immunoprecipitation (ChIP) . For immunofluorescence and immunocytochemistry applications, the recommended dilution ranges from 1:50 to 1:200, although optimal concentrations should be determined for each specific experimental setup . The antibody is particularly valuable for ChIP experiments that aim to identify the genomic locations where histone H4 is acetylated at lysine 12, allowing researchers to correlate this modification with transcriptional activity and chromatin states .

What is the significance of H4K12 acetylation in chromatin biology?

H4K12 acetylation (H4K12ac) is a highly conserved histone modification present from yeast to humans, indicating its fundamental importance in chromatin regulation . This modification is predominantly found on newly synthesized histone H4, where it is catalyzed by the histone acetyltransferase 1 (HAT1) enzyme in complex with RbAp46 . H4K12ac, typically occurring alongside H4K5 acetylation, plays a critical role in several nuclear processes:

• Nucleosome Assembly: H4K12ac is enriched on newly synthesized histones and has been implicated in regulating CAF-1-mediated nucleosome assembly during DNA replication .

• Chromatin Maturation: The modification serves as a mark of newly incorporated histones before being removed during chromatin maturation processes.

• Transcriptional Regulation: While primarily associated with histone deposition, H4K12ac can also influence gene expression patterns in specific genomic contexts.

• DNA Replication: The modification is particularly important during S phase of the cell cycle, where it may help coordinate histone deposition with DNA replication .

How should HIST1H4A (Ab-12) Antibody be stored and handled to maintain its activity?

For optimal performance and longevity of the HIST1H4A (Ab-12) antibody, proper storage and handling are essential. The antibody is typically supplied in liquid format with a preservative such as 0.03% Proclin 300 to prevent microbial contamination . To maintain antibody activity:

• Storage Temperature: Store the antibody at -20°C for long-term storage and at 4°C for short-term use (less than one month).

• Aliquoting: To prevent repeated freeze-thaw cycles, divide the antibody into small, single-use aliquots before freezing. Multiple freeze-thaw cycles can lead to protein denaturation and loss of binding activity.

• Working Dilutions: Prepare working dilutions immediately before use and discard any unused diluted antibody.

• Handling: When handling the antibody, use sterile pipette tips and tubes to prevent contamination.

• Thawing: Thaw frozen antibody aliquots gradually at room temperature or in a refrigerator, avoiding rapid temperature changes that could compromise antibody structure.

Following these guidelines will help ensure consistent experimental results and maximize the usable lifespan of the antibody.

How can HIST1H4A (Ab-12) Antibody be optimized for Chromatin Immunoprecipitation (ChIP) experiments?

Optimizing HIST1H4A (Ab-12) antibody for ChIP experiments requires careful attention to several experimental parameters:

By systematically optimizing these parameters, researchers can achieve high specificity and sensitivity in ChIP experiments using the HIST1H4A (Ab-12) antibody.

What are the technical considerations for Western blot analysis using HIST1H4A (Ab-12) Antibody?

Western blot analysis using HIST1H4A (Ab-12) antibody presents unique challenges due to the small size of histone proteins and the specificity required for detecting acetylated epitopes:

• Sample Preparation:

  • Use specialized histone extraction protocols that preserve post-translational modifications

  • Include histone deacetylase inhibitors (e.g., sodium butyrate, trichostatin A) in lysis buffers

  • Consider using acidic extraction methods for enriching histone proteins

• Gel Electrophoresis:

  • Use high percentage (15-18%) SDS-PAGE gels or specialized Triton-Acid-Urea (TAU) gels for better resolution of histone proteins

  • Include molecular weight markers appropriate for small proteins (histone H4 runs at approximately 11-14 kDa)

  • Consider using gradient gels for better separation of similarly sized histone variants

• Transfer Conditions:

  • Optimize transfer time and voltage for small proteins (typically lower voltage for longer time)

  • PVDF membranes often provide better results than nitrocellulose for histone antibodies

  • Consider using methanol-free transfer buffers for acetylation-specific antibodies

• Blocking and Antibody Incubation:

  • Use 5% BSA rather than milk for blocking and antibody dilution to reduce background

  • Incubate primary antibody at recommended dilutions (typically 1:1000-1:2000) overnight at 4°C

  • Include appropriate positive controls (e.g., recombinant acetylated H4)

• Signal Detection:

  • Use highly sensitive ECL substrates compatible with the detection of low abundance histone modifications

  • Consider fluorescent secondary antibodies for more quantitative analysis

By following these technical considerations, researchers can achieve specific detection of H4K12ac and avoid common pitfalls in histone Western blotting.

How does HAT1-mediated H4K12 acetylation differ between H3.1-H4 and H3.3-H4 complexes?

HAT1-RbAp46 exhibits differential acetylation activity toward H4 depending on whether it is complexed with H3.1 or H3.3, which has important implications for using HIST1H4A (Ab-12) antibody in research contexts:

• Preferential Acetylation of H3.1-H4: HAT1-RbAp46 more efficiently acetylates H4K12 when H4 is in complex with H3.1 compared to H3.3 . This preferential acetylation has been demonstrated both with enzyme purified from human cells and with recombinant HAT1-RbAp46 complex .

• Binding Affinity Differences: Significantly more HAT1 and RbAp46 co-purify with H3.1 than with H3.3, suggesting a higher binding affinity for the H3.1-H4 complex .

• Functional Implications:

  • The differential acetylation may reflect the distinct roles of H3.1 and H3.3 in chromatin assembly and gene regulation

  • H3.1 is predominantly incorporated during DNA replication via CAF-1, while H3.3 is deposited in a replication-independent manner via HIRA

  • The preferential acetylation of H4 in H3.1-H4 complexes may facilitate replication-coupled nucleosome assembly

• Research Applications: When using HIST1H4A (Ab-12) antibody, researchers should consider:

  • Cell cycle stage of analyzed samples (S-phase versus non-dividing cells)

  • Relative abundance of H3.1 versus H3.3 in their experimental system

  • Potential bias in detecting H4K12ac on H3.1-containing versus H3.3-containing nucleosomes

This differential acetylation pattern provides important context for interpreting results obtained with HIST1H4A (Ab-12) antibody and underscores the complex regulation of histone modifications in different nucleosome contexts.

What protocols can be used to validate the specificity of HIST1H4A (Ab-12) Antibody?

Validating antibody specificity is crucial for ensuring reliable experimental results. For HIST1H4A (Ab-12) antibody, several approaches can be employed:

• Peptide Competition Assay:

  • Pre-incubate the antibody with excess acetylated H4K12 peptide (immunogen)

  • In parallel, pre-incubate with unacetylated H4K12 peptide or irrelevant acetylated peptides

  • Compare binding in Western blot or immunofluorescence

  • Specific signal should be blocked by the acetylated H4K12 peptide but not by control peptides

• Knockout/Knockdown Validation:

  • Test the antibody on samples from HAT1 knockout/knockdown cells, which should show reduced H4K12ac levels

  • Compare with wild-type cells to confirm specificity

  • Include rescue experiments by re-expressing HAT1 to restore the signal

• Histone Deacetylase (HDAC) Inhibitor Treatment:

  • Treat cells with HDAC inhibitors to increase global histone acetylation

  • Confirm increased signal with the HIST1H4A (Ab-12) antibody

  • Compare with other acetylation-specific antibodies as positive controls

• Mass Spectrometry Validation:

  • Perform immunoprecipitation with the antibody followed by mass spectrometry

  • Confirm enrichment of peptides containing acetylated H4K12

  • Analyze co-precipitation of other histone modifications to assess cross-reactivity

• Comparison with Other Validated Antibodies:

  • Test multiple antibodies targeting H4K12ac from different suppliers

  • Compare staining patterns across different applications

  • Consistent results across antibodies increase confidence in specificity

By implementing these validation protocols, researchers can ensure that their results with HIST1H4A (Ab-12) antibody accurately reflect the biological distribution of H4K12 acetylation.

How can HIST1H4A (Ab-12) Antibody be used to investigate epigenetic changes during DNA replication?

HIST1H4A (Ab-12) antibody provides a valuable tool for investigating the dynamics of H4K12 acetylation during DNA replication and chromatin assembly:

• Cell Cycle Synchronization Experiments:

  • Synchronize cells using thymidine block, serum starvation, or cell sorting

  • Collect samples at different time points throughout S phase

  • Use HIST1H4A (Ab-12) antibody in Western blot or immunofluorescence to track H4K12ac levels

  • Correlate with markers of DNA replication (e.g., PCNA, EdU incorporation)

• ChIP-seq Analysis Across the Cell Cycle:

  • Perform ChIP-seq with HIST1H4A (Ab-12) antibody in synchronized cell populations

  • Analyze the genome-wide distribution of H4K12ac during different cell cycle phases

  • Identify regions where H4K12ac is enriched during S phase

  • Correlate with replication timing data and other histone marks

• Pulse-Chase Experiments:

  • Label newly synthesized histones (e.g., using SNAP-tag or biotin-based approaches)

  • Track H4K12ac on new versus old histones using HIST1H4A (Ab-12) antibody

  • Determine the kinetics of H4K12ac establishment and removal

• Replication Stress Response:

  • Induce replication stress using hydroxyurea or other agents

  • Monitor changes in H4K12ac patterns using HIST1H4A (Ab-12) antibody

  • Investigate how replication fork stalling affects histone deposition and modification

• Co-localization Studies:

  • Perform dual immunofluorescence with HIST1H4A (Ab-12) antibody and antibodies against:

    • Replication machinery components (PCNA, DNA polymerases)

    • Histone chaperones (CAF-1, ASF1)

    • HAT1 and RbAp46

  • Analyze co-localization at replication forks and newly synthesized DNA

These approaches can provide insights into how H4K12 acetylation contributes to chromatin assembly during DNA replication and how this process differs between normal replication and conditions of replication stress.

What controls should be included when interpreting HIST1H4A (Ab-12) Antibody results?

Proper experimental controls are essential for accurate interpretation of results obtained with HIST1H4A (Ab-12) antibody:

• Positive Controls:

  • Cell lines or tissues known to express high levels of H4K12ac (e.g., proliferating cells in S phase)

  • Samples treated with HDAC inhibitors to increase global histone acetylation

  • Purified acetylated H4 peptides or recombinant acetylated histones

• Negative Controls:

  • Samples treated with HAT1 inhibitors or HAT1 knockdown/knockout cells

  • IgG control from the same species as the primary antibody (rabbit)

  • Secondary antibody-only controls to assess non-specific binding

  • Peptide competition controls (pre-incubation with acetylated H4K12 peptide)

• Specificity Controls:

  • Testing for cross-reactivity with other acetylated lysines on H4 (K5, K8, K16)

  • Comparing signal with other validated H4K12ac antibodies

  • Western blot analysis to confirm appropriate molecular weight (11-14 kDa)

• Technical Controls:

  • Loading controls for Western blot (total H4 or total H3)

  • Tissue/cell morphology assessment for IHC/IF

  • Input controls for IP and ChIP experiments

• Biological Context Controls:

  • Cell cycle stage analysis (S phase versus G1/G2)

  • Comparison between replicating and non-replicating cells

  • Differentiation state comparisons

Including these controls in experimental design and data analysis will help ensure the reliability and interpretability of results obtained with HIST1H4A (Ab-12) antibody.

How do the patterns of H4K12 acetylation differ across various cell types and tissues?

H4K12 acetylation displays distinct patterns across different cell types and tissues, which researchers should consider when designing experiments with HIST1H4A (Ab-12) antibody:

• Proliferative versus Post-mitotic Cells:

  • Proliferating cells generally show higher levels of H4K12ac due to ongoing DNA replication and histone synthesis

  • Post-mitotic cells typically have lower baseline levels but may show specific enrichment at certain genomic loci

• Stem Cell Populations:

  • Embryonic stem cells exhibit distinctive H4K12ac patterns associated with their unique chromatin structure

  • During differentiation, H4K12ac patterns undergo dynamic changes correlating with developmental gene expression programs

• Cancer Cells:

  • Many cancer cell types show altered H4K12ac patterns compared to normal counterparts

  • Changes may reflect dysregulation of HAT1 activity or altered histone turnover rates

  • These alterations can be detected using HIST1H4A (Ab-12) antibody in immunohistochemistry of tumor samples

• Tissue-Specific Patterns:

  • Brain tissue shows region-specific H4K12ac patterns associated with neuronal activity and memory formation

  • Liver exhibits distinctive H4K12ac distribution related to metabolic gene regulation

  • Reproductive tissues demonstrate unique H4K12ac profiles linked to gametogenesis

• Cell Cycle Variation:

  • H4K12ac levels peak during S phase when new histones are being synthesized and incorporated

  • Different genomic regions show distinct dynamics of H4K12ac throughout the cell cycle

Understanding these tissue and cell type-specific variations is crucial for properly interpreting results obtained with HIST1H4A (Ab-12) antibody and for designing appropriate experimental controls.

How can quantitative analysis be performed on HIST1H4A (Ab-12) Antibody signal in imaging applications?

Quantitative analysis of H4K12ac signal using HIST1H4A (Ab-12) antibody in imaging applications requires careful attention to methodological considerations:

• Image Acquisition Parameters:

  • Use consistent exposure times, gain settings, and binning across all samples

  • Avoid saturated pixels that would compromise quantification

  • Capture images at appropriate bit depth (16-bit recommended) to maximize dynamic range

  • Include fluorescence intensity standards when possible

• Background Correction Methods:

  • Implement appropriate background subtraction

  • Use rolling ball algorithm for uneven background

  • Include no-primary-antibody controls to establish baseline

• Nuclear Segmentation Approaches:

  • Use DAPI or other nuclear counterstains for accurate nuclear segmentation

  • Employ automated segmentation algorithms (watershed, thresholding)

  • Validate segmentation accuracy through manual inspection

• Quantification Metrics:

  • Mean nuclear intensity (average H4K12ac signal per nucleus)

  • Integrated nuclear intensity (total H4K12ac signal per nucleus)

  • Coefficient of variation (heterogeneity of H4K12ac distribution)

  • Sub-nuclear distribution patterns (euchromatin versus heterochromatin enrichment)

• Statistical Analysis:

  • Analyze sufficient cell numbers (typically >100 cells per condition)

  • Use appropriate statistical tests based on data distribution

  • Account for biological replicates and technical variation

  • Consider cell cycle stage when interpreting H4K12ac levels

• Visualization Methods:

  • Heatmaps of nuclear H4K12ac intensity across cell populations

  • Violin or box plots showing distribution of H4K12ac levels

  • Correlation plots with other histone marks or cellular features

Implementing these quantitative approaches allows researchers to extract meaningful biological insights from imaging experiments using HIST1H4A (Ab-12) antibody beyond qualitative visual assessment.

What are common challenges when using HIST1H4A (Ab-12) Antibody in different applications?

Researchers may encounter several challenges when working with HIST1H4A (Ab-12) antibody across different experimental platforms:

• Western Blotting Challenges:

  • Weak Signal: Ensure proper histone extraction that preserves acetylation marks

  • Multiple Bands: May indicate cross-reactivity with other acetylated lysines or degradation products

  • High Background: Optimize blocking conditions (5% BSA typically works better than milk)

  • Solution: Use specialized histone extraction protocols and include HDAC inhibitors during sample preparation

• Immunofluorescence/ICC Challenges:

  • Low Signal-to-Noise Ratio: Optimize fixation methods (paraformaldehyde generally works well)

  • Inconsistent Staining: Ensure consistent permeabilization across samples

  • Autofluorescence: Include appropriate quenching steps if needed

  • Solution: Test different antibody concentrations (1:50-1:200) and extend primary antibody incubation time (overnight at 4°C)

• ChIP Challenges:

  • Poor Enrichment: Optimize chromatin fragmentation and increase antibody amount

  • High Background: Increase washing stringency and use appropriate blocking agents

  • Inconsistent Results: Standardize crosslinking and sonication conditions

  • Solution: Include spike-in controls and optimize antibody-to-chromatin ratio

• Immunohistochemistry Challenges:

  • Weak or Absent Staining: Implement antigen retrieval methods (heat-mediated with citrate buffer, pH 6.0)

  • Non-specific Staining: Optimize blocking and increase washing steps

  • Tissue-Dependent Variation: Adjust fixation times based on tissue type

  • Solution: Test different antibody concentrations and antigen retrieval methods

• ELISA Challenges:

  • Poor Sensitivity: Use high-binding plates and optimize coating conditions

  • Cross-Reactivity: Include appropriate competing peptides as controls

  • Non-Linear Standard Curves: Adjust antibody and sample dilutions

  • Solution: Implement sandwich ELISA format for increased specificity

By recognizing and addressing these common challenges, researchers can optimize their experimental protocols for more reliable and reproducible results using HIST1H4A (Ab-12) antibody.

How can HIST1H4A (Ab-12) Antibody be used in multiplexed detection with other histone marks?

Multiplexed detection of H4K12ac alongside other histone modifications provides valuable insights into chromatin regulation. Several approaches can be used with HIST1H4A (Ab-12) antibody:

• Sequential Chromatin Immunoprecipitation (Re-ChIP):

  • Perform initial ChIP with HIST1H4A (Ab-12) antibody

  • Elute under mild conditions that preserve protein-DNA interactions

  • Perform second ChIP with antibody against another histone mark

  • This approach identifies genomic regions with co-occurrence of both modifications

  • Example protocol: First ChIP for H4K12ac, followed by second ChIP for H3K4me3 to identify active promoters with newly deposited histones

• Multicolor Immunofluorescence:

  • Select primary antibodies from different host species (HIST1H4A Ab-12 is rabbit-derived)

  • Use spectrally distinct fluorophore-conjugated secondary antibodies

  • Include appropriate controls for spectral overlap/bleed-through

  • Example setup: HIST1H4A (Ab-12) with anti-rabbit-Alexa488 + mouse anti-H3K9me3 with anti-mouse-Alexa594

• Multiplexed Western Blotting:

  • Strip and reprobe membranes sequentially

  • Use different size markers to distinguish histone marks

  • Alternatively, use fluorescent secondary antibodies with different excitation/emission spectra

  • Example approach: Probe first with HIST1H4A (Ab-12), image, strip, then reprobe with antibody against total H4

• Mass Cytometry (CyTOF):

  • Conjugate HIST1H4A (Ab-12) antibody to a unique metal isotope

  • Combine with other histone mark antibodies conjugated to different metals

  • Analyze single-cell histone modification profiles

  • Provides quantitative data on co-occurrence of multiple histone marks at single-cell resolution

• Sequential Immunohistochemistry:

  • Perform first staining with HIST1H4A (Ab-12) antibody

  • Document results with whole-slide scanning

  • Strip antibodies while preserving tissue architecture

  • Perform second staining with antibody against another histone mark

  • Digitally overlay images to analyze co-localization

These multiplexing approaches enable researchers to study the complex interplay between H4K12ac and other epigenetic modifications across various experimental platforms.

What emerging technologies might enhance research using HIST1H4A (Ab-12) Antibody?

Several cutting-edge technologies are poised to expand the utility of HIST1H4A (Ab-12) antibody in epigenetic research:

• CUT&Tag and CUT&RUN Technologies:

  • These techniques offer improved signal-to-noise ratio compared to traditional ChIP

  • HIST1H4A (Ab-12) antibody could be adapted for these platforms to map H4K12ac genome-wide with higher resolution

  • Potential for single-cell applications to reveal cell-to-cell variation in H4K12ac patterns

• Live-Cell Imaging of Histone Modifications:

  • Development of acetylation-specific intrabodies derived from HIST1H4A (Ab-12)

  • Coupling with FRET-based sensors to monitor H4K12ac dynamics in real-time

  • Integration with lattice light-sheet microscopy for high-resolution 3D imaging of H4K12ac in living cells

• Spatial Transcriptomics Integration:

  • Combining HIST1H4A (Ab-12) immunofluorescence with spatial transcriptomics

  • Correlating H4K12ac patterns with gene expression in intact tissues

  • Mapping the relationship between histone modifications and cellular heterogeneity

• Nanobody Development:

  • Engineering H4K12ac-specific nanobodies based on HIST1H4A (Ab-12) epitope recognition

  • Smaller size allows better access to densely packed chromatin regions

  • Potential for improved ChIP efficiency and novel super-resolution imaging applications

• Multi-omic Single-Cell Technologies:

  • Integration of HIST1H4A (Ab-12) antibody into protocols for simultaneous profiling of:

    • Histone modifications (including H4K12ac)

    • Transcriptome

    • Chromatin accessibility

    • Nuclear protein levels

  • Provides comprehensive view of how H4K12ac relates to other cellular parameters

These emerging technologies promise to extend the capabilities of HIST1H4A (Ab-12) antibody and provide deeper insights into the biological roles of H4K12 acetylation in chromatin regulation and gene expression.

How is our understanding of H4K12 acetylation evolving in the context of disease and development?

Research using tools like HIST1H4A (Ab-12) antibody is advancing our understanding of H4K12 acetylation in various pathological and developmental contexts:

• Neurodegenerative Diseases:

  • Altered H4K12ac patterns have been observed in models of Alzheimer's disease

  • Age-associated decline in H4K12ac correlates with memory impairment

  • HIST1H4A (Ab-12) antibody could help identify therapeutic targets for cognitive enhancement

• Cancer Epigenetics:

  • Dysregulation of HAT1 activity and H4K12ac patterns in various cancer types

  • Potential prognostic significance of H4K12ac distribution in tumor samples

  • Opportunities for developing epigenetic biomarkers using HIST1H4A (Ab-12) antibody in immunohistochemistry

• Developmental Reprogramming:

  • Dynamic changes in H4K12ac during cellular differentiation and development

  • Role in establishing and maintaining cell-type-specific gene expression programs

  • Potential involvement in epigenetic memory during development

• Aging Processes:

  • Global and gene-specific changes in H4K12ac with advancing age

  • Correlation with transcriptional changes in aging tissues

  • Potential target for interventions aimed at healthy aging

• Reproductive Biology:

  • Distinct patterns of H4K12ac in gametes and early embryos

  • Potential role in transgenerational epigenetic inheritance

  • Implications for assisted reproductive technologies

Future research using HIST1H4A (Ab-12) antibody will likely continue to expand our understanding of these disease and developmental contexts, potentially leading to new diagnostic approaches and therapeutic strategies targeting H4K12 acetylation or the enzymes that regulate it.