HIST1H4A (Ab-77) Antibody

What is the HIST1H4A protein and why is it significant in research?

HIST1H4A (Histone Cluster 1, H4a) is one of several genes encoding histone H4, a core component of the nucleosome structure in chromatin. This protein plays a crucial role in DNA packaging, chromatin structure, and epigenetic regulation of gene expression. Histone H4 is highly conserved and subject to various post-translational modifications that influence chromatin dynamics and transcriptional activity. Research on HIST1H4A is particularly significant in epigenetics, cancer biology, and developmental studies where histone modifications serve as critical regulatory mechanisms . The protein has multiple aliases including H4/A, H4FA, and others, reflecting the conservation and importance of this histone variant across multiple genomic loci .

What are the key specifications of the HIST1H4A (Ab-77) Antibody?

The HIST1H4A (Ab-77) Antibody is a polyclonal antibody raised in rabbits against a peptide sequence around the Lysine 77 site derived from human Histone H4 . Its key specifications include:

This antibody has been specifically designed to recognize the lysine residue at position 77 of human Histone H4, making it a valuable tool for studying this specific region of the protein .

How does the HIST1H4A (Ab-77) Antibody differ from other H4 histone antibodies?

The HIST1H4A (Ab-77) Antibody differs from other H4 histone antibodies primarily in its specific targeting of the lysine residue at position 77. While other antibodies may target different regions or modifications of Histone H4, this antibody provides researchers with a tool to specifically study this region . For comparison:

• The antibody described in result (ABIN7139153) targets acetylated lysine 12 (acLys12) of Histone H4.

• Other antibodies may target different modifications like methylated lysine 20 (meLys20) or acetylated lysine sites such as acLys8, acLys16, acLys31, or acLys56 .

• Some antibodies, like the Histone H4 (L64C1) Mouse mAb (result ), target the amino-terminal sequence of human histone H4 rather than specific modification sites.

These differences are important for experimental design, as each antibody provides insights into different aspects of histone biology and chromatin regulation .

What are the validated applications for HIST1H4A (Ab-77) Antibody in chromatin research?

The HIST1H4A (Ab-77) Antibody has been validated for several important chromatin research applications:

• Immunohistochemistry (IHC): The antibody has been tested for paraffin-embedded tissue sections with recommended dilutions of 1:1-1:10. Validation data shows successful staining in human placenta and small intestine tissues using a Leica Bond system with high-pressure citrate buffer (pH 6.0) antigen retrieval .

• Immunofluorescence (IF): The antibody has been validated for cellular localization studies with recommended dilutions of 1:1-1:10. Testing in HeLa cells has demonstrated its efficacy for nuclear protein detection .

• ELISA: The antibody has been validated for enzyme-linked immunosorbent assays, allowing for quantitative detection of HIST1H4A protein .

While not explicitly validated for this antibody, other HIST1H4A antibodies have been successfully used in:

• Chromatin Immunoprecipitation (ChIP): Important for studying histone-DNA interactions and genomic occupancy .

• Western Blotting: For detecting HIST1H4A protein levels in cell or tissue lysates .

Each application requires specific optimization of protocols, including proper sample preparation, antibody dilution, and detection systems appropriate for the research question being addressed.

How should I optimize the HIST1H4A (Ab-77) Antibody for immunohistochemistry experiments?

Optimizing the HIST1H4A (Ab-77) Antibody for immunohistochemistry requires careful attention to several key parameters:

• Antigen Retrieval: Based on validation data, high-pressure antigen retrieval in citrate buffer (pH 6.0) has proven effective. This step is critical for exposing epitopes that may be masked during fixation .

• Blocking: Use 10% normal goat serum for 30 minutes at room temperature to reduce non-specific binding. This blocking step is important before primary antibody application .

• Primary Antibody Dilution: Start with the recommended dilution of 1:1-1:10 in 1% BSA. The optimal dilution may vary depending on tissue type and fixation method .

• Incubation Conditions: Incubate the primary antibody overnight at 4°C for optimal binding .

• Detection System: A biotinylated secondary antibody followed by an HRP-conjugated SP system has been validated for visualization .

• Controls: Always include appropriate positive and negative controls:

  • Positive control: Human placenta or small intestine tissues

  • Negative control: Primary antibody omission or isotype control

• Optimization Strategy: If initial results are suboptimal, consider a titration experiment with different antibody dilutions (e.g., 1:1, 1:5, 1:10) and varying antigen retrieval conditions.

The published validation images demonstrate successful staining of nuclear histones in human placenta and small intestine tissues, which can serve as reference patterns for your experiments .

What are the critical considerations for using HIST1H4A antibodies in ChIP experiments?

While the HIST1H4A (Ab-77) Antibody specifically hasn't been extensively documented for ChIP, several critical considerations apply when using HIST1H4A antibodies in ChIP experiments:

• Crosslinking Optimization: The standard 1% formaldehyde for 10 minutes at room temperature may need adjustment. Histones often require milder crosslinking conditions (0.5-0.75% formaldehyde for 5-8 minutes) to prevent epitope masking.

• Sonication Parameters: Chromatin fragmentation to 200-500bp is optimal for histone ChIP. Over-sonication can destroy epitopes while under-sonication results in poor resolution.

• Antibody Amount: Typically 2-5μg of antibody per ChIP reaction is recommended, but titration experiments are advisable to determine optimal amounts for each experimental system .

• Controls:

  • Input control (pre-immunoprecipitation chromatin)

  • IgG control (non-specific rabbit IgG)

  • Positive control regions (known targets of histone H4)

  • Negative control regions (genomic regions devoid of histone H4 modifications)

• Washing Stringency: Histone ChIP often requires more stringent washing conditions than transcription factor ChIP to minimize background.

• Validation Strategy: qPCR analysis of known target regions or genome-wide sequencing (ChIP-seq) with appropriate bioinformatic analysis to verify specificity.

• Multiplexing Considerations: When examining multiple histone modifications, sequential ChIP (re-ChIP) may be necessary to determine co-occurrence on the same DNA fragments.

For HIST1H4A antibodies specifically targeting lysine 77, special attention should be paid to potential epitope masking by adjacent modifications or protein interactions that might affect antibody accessibility in the chromatin context .

How can I verify the specificity of the HIST1H4A (Ab-77) Antibody?

Verifying antibody specificity is crucial for reliable research outcomes. For the HIST1H4A (Ab-77) Antibody, consider these verification approaches:

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide (sequence around Lys77 of human Histone H4) before application to samples. Signal reduction confirms specificity.

• Western Blot Analysis: Though not explicitly listed as a validated application for this antibody, western blot can verify specificity by confirming a single band at the expected molecular weight of approximately 11 kDa (the known size of histone H4) .

• Knockout/Knockdown Controls: While complete histone H4 knockout is lethal, RNAi approaches targeting specific H4 variants can provide partial validation when combined with other methods.

• Cross-Reactivity Testing: Test the antibody on samples from different species. Since this antibody is designed for human samples, specificity would be indicated by reduced or absent signal in non-human samples (unless the epitope is fully conserved) .

• Multiple Antibody Validation: Compare results with other antibodies targeting different epitopes of the same protein. Correlation of signals suggests specificity.

• Mass Spectrometry Validation: For the highest level of validation, immunoprecipitation followed by mass spectrometry can identify all proteins pulled down by the antibody.

• Consultation of Antibody Validation Databases: Resources like the recently developed interactive database for histone antibody specificity can provide valuable information about cross-reactivity profiles .

Remember that specificity verification should be performed in the context of your particular experimental system and application.

What are common problems with HIST1H4A antibody experiments and how can I address them?

Researchers frequently encounter several challenges when working with histone antibodies including the HIST1H4A (Ab-77) Antibody:

• High Background in Immunostaining:

  • Problem: Diffuse or non-nuclear staining

  • Solutions:

    • Increase blocking time/concentration (try 5% BSA instead of 1%)

    • Use more stringent washing (increase wash buffer volume and duration)

    • Optimize primary antibody dilution (try higher dilutions)

    • Ensure proper fixation techniques (overfixation can increase background)

• Weak or No Signal:

  • Problem: Insufficient antibody binding or detection

  • Solutions:

    • Enhance antigen retrieval (increase time or try different buffers)

    • Decrease antibody dilution (use more concentrated antibody)

    • Extend primary antibody incubation (try 48 hours at 4°C instead of overnight)

    • Check sample preparation (ensure proper fixation preserves epitopes)

    • Verify storage conditions (antibody activity may decrease with improper storage)

• Cross-Reactivity Issues:

  • Problem: Antibody binds to unintended targets

  • Solutions:

    • Perform peptide competition assays to verify specificity

    • Use more stringent washing conditions

    • Pre-adsorb antibody with potential cross-reactive proteins

• Inconsistent Results Between Experiments:

  • Problem: Variable staining patterns or intensities

  • Solutions:

    • Standardize all protocol steps (timing, temperatures, reagent concentrations)

    • Use positive controls in each experiment

    • Prepare larger batches of working dilutions

    • Consider automated staining platforms for greater consistency

• Epitope Masking by Adjacent Modifications:

  • Problem: Nearby post-translational modifications affect antibody binding

  • Solutions:

    • Use antibodies that are insensitive to adjacent modifications

    • Perform parallel experiments with antibodies recognizing different epitopes

    • Consider mass spectrometry-based approaches for comprehensive modification analysis

Consulting the documentation provided with the antibody and reaching out to the manufacturer's technical support can provide additional troubleshooting guidance specific to this particular antibody .

How do storage conditions and handling affect HIST1H4A (Ab-77) Antibody performance?

Proper storage and handling are critical for maintaining antibody performance over time:

Following these storage and handling guidelines will help ensure optimal antibody performance throughout your research project .

How can HIST1H4A (Ab-77) Antibody be used to study epigenetic modifications in cancer research?

The HIST1H4A (Ab-77) Antibody offers valuable opportunities for investigating epigenetic modifications in cancer research:

• Mapping Histone Modification Landscapes:

  • This antibody can be used to examine the distribution of H4 proteins across cancer genomes when used in ChIP-seq experiments.

  • Changes in histone H4 occupancy or modifications at Lys77 and surrounding regions can be correlated with gene expression alterations in cancer cells.

• Tissue Microarray Analysis:

  • The validated IHC application (dilution 1:1-1:10) enables screening of multiple cancer tissue samples simultaneously .

  • This approach can identify correlations between histone patterns and clinical parameters (tumor grade, stage, patient outcome).

  • The successful staining in human placenta and small intestine tissues demonstrates the antibody's utility in human tissue analysis .

• Cell Line Models:

  • Immunofluorescence applications in cancer cell lines can reveal changes in nuclear organization and histone distribution during cancer progression.

  • The documented effectiveness in HeLa cells (a cervical cancer cell line) supports this application .

• Drug Response Studies:

  • Monitor changes in histone H4 status in response to epigenetic drugs (HDAC inhibitors, HAT inhibitors).

  • Compare pre- and post-treatment samples to assess therapeutic effects on histone modifications.

• Multi-parameter Analysis:

  • Combine with antibodies targeting other histone modifications in multiplex immunofluorescence to create comprehensive epigenetic profiles.

  • Correlate findings with DNA methylation data for integrated epigenetic analysis.

• Circulating Tumor Cell (CTC) Analysis:

  • Apply immunocytochemistry protocols to analyze histone H4 status in CTCs isolated from patient blood samples.

  • This may provide diagnostic or prognostic information without invasive biopsies.

For these applications, researchers should establish appropriate positive and negative controls specific to their cancer model systems, and consider combining the HIST1H4A (Ab-77) Antibody with other epigenetic markers for comprehensive analysis .

What are the considerations for using HIST1H4A antibodies in multiplexed immunofluorescence studies?

Multiplexed immunofluorescence with HIST1H4A antibodies requires careful planning and optimization:

• Antibody Source Compatibility:

  • When combining multiple antibodies, select primary antibodies raised in different host species to avoid cross-reactivity of secondary antibodies.

  • If using multiple rabbit antibodies (like the HIST1H4A (Ab-77) Antibody), consider sequential staining with direct labeling or tyramide signal amplification (TSA) approaches .

• Spectral Overlap Considerations:

  • Choose fluorophores with minimal spectral overlap for secondary antibodies.

  • Common combinations include FITC/Alexa 488 (green), TRITC/Alexa 555 (red), and DAPI (blue) for nuclear counterstaining.

  • For advanced multiplexing, consider spectral imaging systems that can separate overlapping fluorophore signals.

• Antibody Validation for Multiplexing:

  • Test each antibody individually before combining them.

  • Perform staining controls to ensure that the presence of one antibody doesn't interfere with the binding of others.

  • Include single-stain controls for accurate compensation in analysis.

• Optimization of Staining Sequence:

  • The order of antibody application can significantly impact results.

  • For sequential staining, typically begin with the weakest signal antibody and end with the strongest.

  • When examining multiple histone modifications, consider their abundance and epitope accessibility.

• Image Acquisition Parameters:

  • Standardize exposure times and gain settings.

  • Use appropriate filters to minimize bleed-through between channels.

  • Consider confocal microscopy for improved signal separation and subcellular localization.

• Quantitative Analysis Approaches:

  • Develop consistent quantification strategies (nuclear intensity, foci counting).

  • Use appropriate software for colocalization analysis.

  • Consider machine learning approaches for pattern recognition in complex datasets.

• Protocol Adjustments for HIST1H4A (Ab-77):

  • The recommended dilution for IF (1:1-1:10) may need adjustment in multiplexed settings .

  • Validate that the relatively high antibody concentration doesn't create background issues in multiplexed experiments.

  • Consider signal amplification methods if working with low-abundance modifications.

These considerations will help ensure reliable and interpretable results when incorporating HIST1H4A antibodies into complex multiplexed immunofluorescence studies .

How can I integrate HIST1H4A antibody data with other omics approaches for comprehensive epigenetic analysis?

Integrating HIST1H4A antibody data with other omics approaches creates a more comprehensive understanding of epigenetic regulation:

• ChIP-seq Integration Strategies:

  • With RNA-seq: Correlate histone H4 occupancy/modifications with gene expression patterns to identify regulatory relationships.

  • With ATAC-seq: Compare chromatin accessibility data with histone H4 distribution to understand chromatin structure dynamics.

  • With DNA Methylation Arrays/WGBS: Analyze the relationship between histone H4 status and DNA methylation patterns at specific genomic regions.

• Multi-omics Data Analysis Frameworks:

  • Use integrated analysis platforms like Galaxy, Bioconductor, or specialized epigenomics tools.

  • Apply machine learning approaches to identify complex patterns across different data types.

  • Consider trajectory analyses to understand temporal dynamics of epigenetic changes.

• Visualization and Integration Tools:

  • Genome browsers (UCSC, IGV) with multiple tracks displaying different omics data types.

  • HiC/3D genome data visualization to correlate histone marks with chromatin architecture.

  • Network analysis tools to identify regulatory hubs where histone H4 plays a critical role.

• Statistical Approaches for Integration:

  • Correlation analyses (Pearson, Spearman) between histone H4 signals and other omics features.

  • Dimension reduction techniques (PCA, t-SNE, UMAP) for visualizing multi-dimensional epigenetic landscapes.

  • Clustering methods to identify regions with similar epigenetic profiles across multiple datasets.

• Functional Validation Strategies:

  • CRISPR-based approaches to modify specific histone residues or regulatory elements.

  • Reporter assays to test the functional impact of regions with specific histone H4 modifications.

  • Pharmacological interventions targeting histone-modifying enzymes to validate causal relationships.

• Sample Matching Considerations:

  • Whenever possible, generate multiple omics datasets from the same samples.

  • For public data integration, carefully match experimental conditions, cell types, and processing methods.

  • Consider batch effects and appropriate normalization methods when combining datasets.

• Practical Workflow Example:

  • Perform ChIP-seq with HIST1H4A antibody and RNA-seq on the same samples.

  • Identify genomic regions with significant histone H4 occupancy/modifications.

  • Correlate these regions with gene expression changes.

  • Validate key findings with targeted experiments (qPCR, reporter assays).

  • Extend insights with public database integration (ENCODE, Roadmap Epigenomics).

What are the latest findings regarding HIST1H4A modifications in development and disease?

Recent research has revealed several important findings regarding HIST1H4A modifications in development and disease:

• Cancer Biology Insights:

  • Altered patterns of histone H4 modifications, particularly acetylation and methylation, have been associated with various cancer types and may serve as potential biomarkers or therapeutic targets.

  • Changes in enzymes that modify histone H4, such as histone deacetylases (HDACs) and histone acetyltransferases (HATs), are frequently observed in cancer, making them important drug targets.

• Developmental Regulation:

  • Histone H4 modifications play crucial roles in embryonic development, with specific patterns associated with cell differentiation and lineage commitment.

  • Dynamic changes in H4 modification patterns help regulate gene expression during critical developmental windows.

• Neurodegenerative Diseases:

  • Emerging evidence suggests that histone H4 modifications may be altered in neurodegenerative conditions like Alzheimer's and Parkinson's diseases.

  • These epigenetic changes may contribute to abnormal gene expression patterns observed in diseased neurons.

• Aging Research:

  • Global changes in histone H4 modification patterns occur during normal aging processes.

  • These age-related epigenetic alterations may contribute to functional decline and increased disease susceptibility.

• Immune System Regulation:

  • Histone H4 modifications help coordinate immune cell development and function.

  • Dysregulation of these modifications has been implicated in autoimmune disorders and inflammatory conditions.

• Environmental Response Mechanisms:

  • Histone H4 modifications serve as mediators between environmental exposures and gene expression changes.

  • Studies have shown that factors like stress, diet, and toxin exposure can alter histone H4 modification patterns.

• Therapeutic Targeting Approaches:

  • Novel drugs targeting specific histone H4 modifying enzymes are in development for various diseases.

  • Understanding the precise role of different H4 modifications is critical for designing targeted epigenetic therapies.

These findings underscore the importance of tools like the HIST1H4A (Ab-77) Antibody in advancing our understanding of histone biology in both normal development and disease states .

How is single-cell technology changing our understanding of histone H4 dynamics?

Single-cell technologies are revolutionizing our understanding of histone H4 dynamics in several important ways:

• Cellular Heterogeneity Insights:

  • Traditional bulk approaches average histone modification signals across populations, potentially missing critical cell-to-cell variations.

  • Single-cell approaches reveal previously undetectable subpopulations with distinct histone H4 modification patterns.

  • This heterogeneity may explain differential responses to treatments targeting histone modifiers.

• Technological Adaptations for Histone Research:

  • Single-cell ChIP-seq (scChIP-seq): Adaptations of ChIP protocols for minute amounts of starting material allow mapping of histone H4 modifications in individual cells.

  • CUT&Tag/CUT&RUN at single-cell level: These newer techniques offer improved sensitivity for histone modification profiling with reduced background.

  • Single-cell ATAC-seq with histone antibodies: Combining accessibility data with histone information provides multi-dimensional insights.

• Temporal Dynamics Visualization:

  • Single-cell approaches enable reconstruction of epigenetic trajectories during processes like differentiation.

  • Time-course single-cell studies reveal the order and interdependence of different histone H4 modifications.

  • These temporal insights are particularly valuable for understanding developmental processes and disease progression.

• Spatial Context Integration:

  • Emerging spatial technologies allow correlation of histone H4 patterns with cellular location within tissues.

  • This spatial information helps understand how microenvironment influences epigenetic states.

  • Techniques like imaging mass cytometry can be adapted to visualize histone modifications in tissue context.

• Multi-modal Analysis Capabilities:

  • Combined measurement of histone modifications with transcriptome or proteome at single-cell resolution.

  • These integrated approaches help establish direct relationships between histone H4 status and functional outcomes.

  • Methods like CITE-seq principles could potentially be adapted for simultaneous protein and histone modification detection.

• Computational Challenges and Solutions:

  • Development of specialized algorithms to handle sparse data typical of single-cell histone modification profiles.

  • Machine learning approaches for pattern recognition across heterogeneous cell populations.

  • Trajectory inference methods adapted specifically for epigenetic data.

• Technical Considerations for Antibodies:

  • Single-cell applications place additional demands on antibody specificity and sensitivity.

  • Low cell numbers require highly specific antibodies like HIST1H4A (Ab-77) with minimal background binding.

  • Antibody validation becomes even more critical in the single-cell context where false positives can significantly impact interpretations.

These advances are transforming histone research from population-averaged snapshots to dynamic, heterogeneous landscapes with unprecedented resolution .

What are the most effective combinations of histone antibodies for comprehensive epigenetic profiling?

Designing effective antibody panels for comprehensive epigenetic profiling requires strategic combinations that provide complementary information:

• Core Histone Modification Panel:

  • Activating Marks: H3K4me3 (promoters), H3K27ac (enhancers), H3K36me3 (transcribed regions)

  • Repressive Marks: H3K27me3 (Polycomb repression), H3K9me3 (heterochromatin)

  • Histone H4 Modifications: Antibodies like HIST1H4A (Ab-77) and H4K12ac, H4K16ac

  • This combination provides a balanced view of active and repressive chromatin states

• Functional Domain-Specific Combinations:

  • Promoter Analysis: H3K4me3, H3K27me3, H3K9ac, H4K12ac

  • Enhancer Mapping: H3K4me1, H3K27ac, H3K9ac, H4K16ac

  • Heterochromatin Studies: H3K9me3, H4K20me3, HP1α, H4 (general)

  • Transcription Elongation: H3K36me3, H3K79me2, H4K12ac, RNA Pol II

• Cell Type-Specific Optimization:

  • Stem Cell Studies: Add H3K4me1, H3K9me3 (bivalent domains are important)

  • Cancer Research: Include H4K16ac, H4K20me3 (frequently altered in cancer)

  • Neuronal Studies: H3K4me3, H3K27me3, H4K12ac (important for memory formation)

  • Immune Cell Analysis: H3K27ac, H3K4me1, H3K4me3, H4K5ac

• Technical Compatibility Considerations:

  • Select antibodies from different host species when possible

  • If using same-species antibodies, consider sequential staining protocols

  • Verify that antibody combinations don't interfere with each other's binding

  • Test for epitope masking when studying adjacent modifications

• Quantitative Balance Strategy:

  • Include abundant modifications (e.g., H3K4me3 at promoters)

  • Include rarer modifications that may be functionally significant

  • Consider the dynamic range of detection methods

  • Balance broad distribution marks with focal/specific marks

• Validation Approach:

  • Include pan-histone antibodies (like general H3 or H4) as technical controls

  • Use antibodies with established specificity profiles from resources like the interactive histone antibody database

  • Validate novel combinations in well-characterized model systems before applying to experimental samples

• Emerging Combination Trends:

  • Including histone variants (H3.3, H2A.Z) alongside modifications

  • Combining histone marks with chromatin remodelers or readers

  • Adding DNA modification antibodies (5mC, 5hmC) for comprehensive epigenetic view

  • Integrating non-histone chromatin proteins (CTCF, cohesin) for structural context

This strategic approach to antibody combination ensures comprehensive coverage of the epigenetic landscape while maximizing the information gained from precious samples .