HIST1H4A (Ab-20) Antibody

What is HIST1H4A and what role does it play in chromatin biology?

HIST1H4A is one of the core histone proteins that forms nucleosomes, the basic units of chromatin. As a component of nucleosome, HIST1H4A helps wrap and compact DNA, controlling DNA accessibility to cellular machinery. This regulatory role is crucial for transcription regulation, DNA repair, DNA replication, and maintaining chromosomal stability . The accessibility of DNA is regulated through complex post-translational modifications of histones (often referred to as the "histone code") and nucleosome remodeling processes, with HIST1H4A serving as an essential scaffold in this regulatory network .

How is the HIST1H4A (Ab-20) Antibody generated and what epitope does it target?

The HIST1H4A (Ab-20) Antibody is a polyclonal antibody generated in rabbits using a synthetic peptide immunogen derived from human Histone H4 protein. Specifically, this antibody targets the region around lysine 20 (the "Ab-20" designation refers to this target site) . The antibody is generated through immunization of rabbits with the synthetic peptide sequence and subsequently purified using antigen affinity purification methods to ensure specificity . This results in an antibody preparation that specifically recognizes endogenous levels of HIST1H4A protein with modifications at the Lys20 position .

What are the common applications for HIST1H4A (Ab-20) Antibody in research?

The HIST1H4A (Ab-20) Antibody has been validated for multiple research applications, including:

These applications enable researchers to investigate histone modifications, epigenetic regulation, and chromatin dynamics in various experimental contexts .

What are the optimal conditions for using HIST1H4A (Ab-20) Antibody in ChIP experiments?

When designing ChIP experiments with HIST1H4A (Ab-20) Antibody, several key parameters require optimization:

Validation of ChIP efficiency should include both positive and negative control regions to confirm specificity of the antibody binding and precipitation.

How should I optimize immunofluorescence protocols using HIST1H4A (Ab-20) Antibody?

For optimal immunofluorescence results with HIST1H4A (Ab-20) Antibody, consider the following methodology:

• Fixation: Use 4% paraformaldehyde for 15-20 minutes at room temperature, as overfixation can mask the epitope.

• Permeabilization: A brief treatment with 0.2% Triton X-100 for 5-10 minutes is generally effective for nuclear antigens.

• Blocking: Use 5% normal serum (from the species of the secondary antibody) with 0.3% Triton X-100 in PBS for 1 hour at room temperature.

• Antibody dilution: Start with a 1:1 to 1:10 dilution as recommended for IF applications . This relatively concentrated application reflects the need for sufficient binding in fixed tissue contexts.

• Incubation: Overnight incubation at 4°C in a humidified chamber typically yields optimal staining.

• Signal amplification: For weak signals, consider using a biotin-streptavidin system or tyramide signal amplification.

• Controls: Include both primary antibody omission controls and competing peptide controls to validate specificity .

What are the key considerations when using HIST1H4A (Ab-20) Antibody in fixed versus frozen tissue samples?

The performance of HIST1H4A (Ab-20) Antibody differs between fixed and frozen tissue samples:

Fixed tissue considerations:

• Antigen retrieval is critical: Heat-induced epitope retrieval (HIER) using citrate buffer (pH 6.0) or Tris-EDTA buffer (pH 9.0) for 15-20 minutes is often necessary to expose the lysine 20 epitope.

• Paraffin-embedded samples require complete deparaffinization and rehydration.

• Recommended dilution for IHC applications in fixed tissues is 1:20-1:200 .

• Endogenous peroxidase quenching (3% H₂O₂ for 10 minutes) is essential for peroxidase-based detection systems.

Frozen tissue considerations:

• Fixation post-sectioning with cold acetone or 4% PFA is recommended.

• Less aggressive antigen retrieval is typically needed compared to FFPE samples.

• Higher antibody dilutions can often be used due to better epitope preservation.

• Background staining may be more pronounced in frozen sections and may require additional blocking steps.

For both sample types, validation through appropriate positive and negative controls is essential to determine optimal staining conditions .

How can HIST1H4A (Ab-20) Antibody be used to investigate the relationship between H4K20 methylation and DNA damage response?

H4K20 methylation is implicated in DNA damage response pathways, making the HIST1H4A (Ab-20) Antibody valuable for investigating these processes. A methodological approach would include:

• ChIP-seq analysis:

  • Perform ChIP-seq using HIST1H4A (Ab-20) Antibody in control and DNA damage-induced conditions.

  • Compare genome-wide distribution of H4K20 modifications before and after damage induction.

  • Analyze enrichment at known DNA repair hotspots and damage-responsive elements.

• Co-immunoprecipitation studies:

  • Use HIST1H4A (Ab-20) Antibody to pull down H4K20-modified histones.

  • Identify DNA damage response proteins that associate with this modification using mass spectrometry.

  • Validate interactions through reciprocal IP and western blotting.

• Fluorescence recovery after photobleaching (FRAP):

  • Combine immunofluorescence using HIST1H4A (Ab-20) Antibody with live-cell imaging.

  • Measure dynamics of H4K20-modified chromatin regions following DNA damage.

  • Quantify recruitment of repair factors to these regions over time.

• ChIP-qPCR time course experiments:

  • Track changes in H4K20 modification at specific genomic loci during DNA damage repair.

  • Correlate these changes with recruitment of repair factors and resolution of damage .

What are the best approaches for multiplexing HIST1H4A (Ab-20) Antibody with other histone modification antibodies?

Multiplexing histone modification antibodies allows simultaneous examination of multiple epigenetic marks. For HIST1H4A (Ab-20) Antibody, consider these methodological approaches:

• Sequential ChIP (Re-ChIP):

  • Perform first ChIP with HIST1H4A (Ab-20) Antibody.

  • Elute the chromatin complexes under mild conditions.

  • Perform second ChIP with antibodies against other histone modifications.

  • This approach reveals co-occurrence of modifications on the same nucleosomes.

• Multicolor immunofluorescence:

  • Use primary antibodies from different host species (HIST1H4A Ab-20 is rabbit-derived) .

  • Apply species-specific secondary antibodies with distinct fluorophores.

  • Include appropriate controls to rule out cross-reactivity.

  • Analyze co-localization using confocal microscopy and quantitative co-localization metrics.

• Combinatorial indexed ChIP-seq (Co-ChIP):

  • Perform parallel ChIPs with HIST1H4A (Ab-20) and other modification-specific antibodies.

  • Use barcoded adapters to identify chromatin fragments pulled down by each antibody.

  • Computational analysis can identify genomic regions with overlapping or mutually exclusive modification patterns.

• Mass cytometry (CyTOF) for epigenetic profiling:

  • Conjugate HIST1H4A (Ab-20) and other antibodies to distinct metal isotopes.

  • Analyze single-cell epigenetic states across heterogeneous populations.

  • This approach requires careful validation of antibody performance after metal conjugation .

How can HIST1H4A (Ab-20) Antibody be employed in studying the dynamics of histone modifications during cell cycle progression?

Cell cycle-dependent changes in histone modifications are crucial for proper chromatin condensation and segregation. A comprehensive experimental approach using HIST1H4A (Ab-20) Antibody would include:

• Cell synchronization and time-course analysis:

  • Synchronize cells using methods appropriate for your cell type (double thymidine block, nocodazole arrest, etc.).

  • Collect cells at defined time points spanning the cell cycle.

  • Perform ChIP-seq or ChIP-qPCR with HIST1H4A (Ab-20) Antibody at each time point.

  • Analyze dynamics of H4K20 modifications at replication origins, centromeres, and other cell cycle-regulated loci.

• Combined immunofluorescence and flow cytometry:

  • Stain cells with HIST1H4A (Ab-20) Antibody and DNA content marker (propidium iodide or DAPI).

  • Analyze by flow cytometry to quantify H4K20 modification levels in G1, S, G2, and M phases.

  • Use imaging flow cytometry to visualize subcellular localization changes throughout the cell cycle.

• Live-cell imaging with cell cycle markers:

  • Co-transfect cells with fluorescently tagged cell cycle markers (PCNA for S phase, etc.).

  • Perform immunofluorescence with HIST1H4A (Ab-20) Antibody after fixation at specific time points.

  • Track changes in H4K20 modification patterns relative to cell cycle progression markers .

What are the most common causes of non-specific binding with HIST1H4A (Ab-20) Antibody and how can they be mitigated?

Non-specific binding can compromise experimental results when using HIST1H4A (Ab-20) Antibody. Common causes and mitigation strategies include:

• Insufficient blocking:

  • Increase blocking time from 1 hour to 2 hours or overnight at 4°C.

  • Use 5% BSA instead of normal serum for blocking.

  • Add 0.1-0.3% Triton X-100 to reduce hydrophobic interactions.

• Cross-reactivity with similar histone epitopes:

  • Perform peptide competition assays using the immunogenic peptide.

  • Use more stringent washing conditions (increase salt concentration or detergent).

  • Validate results with a second antibody targeting a different epitope on HIST1H4A.

• Overfixation masking epitopes:

  • Optimize fixation time and conditions for your specific sample type.

  • For challenging samples, try alternative fixatives like methanol or acetone.

  • Implement more aggressive antigen retrieval methods if necessary.

• Antibody concentration too high:

  • Titrate the antibody more carefully, testing dilutions from 1:20 to 1:200 .

  • Use a smaller amount of antibody (2-3μg instead of 5μg) in ChIP experiments.

• Sample protein degradation:

  • Add protease inhibitors freshly before sample preparation.

  • Keep samples cold throughout processing.

  • Minimize the time between sample collection and fixation/processing .

How can I validate the specificity of HIST1H4A (Ab-20) Antibody in my experimental system?

Rigorous validation of antibody specificity is crucial for reliable results. For HIST1H4A (Ab-20) Antibody, implement these validation methods:

• Peptide competition assay:

  • Pre-incubate the antibody with excess immunizing peptide (>10-fold molar excess).

  • Perform your experiment with both blocked and unblocked antibody.

  • Specific signals should disappear in the peptide-blocked sample.

• Knockout/knockdown validation:

  • Generate HIST1H4A knockout or knockdown systems using CRISPR/Cas9 or RNAi.

  • Compare staining patterns between wildtype and knockout/knockdown samples.

  • Specific signals should be significantly reduced in the knockout/knockdown samples.

• Orthogonal antibody comparison:

  • Test multiple antibodies targeting different epitopes of HIST1H4A.

  • Compare staining patterns and enrichment profiles.

  • Consistent results across different antibodies suggest specific detection.

• Mass spectrometry validation:

  • Perform immunoprecipitation with HIST1H4A (Ab-20) Antibody.

  • Analyze precipitated proteins by mass spectrometry.

  • Confirm enrichment of HIST1H4A and associated proteins with minimal contaminants .

What are the appropriate positive and negative controls for ChIP experiments using HIST1H4A (Ab-20) Antibody?

Proper controls are essential for interpreting ChIP results correctly:

Positive controls:

• Known enriched genomic regions: Target qPCR primers to genomic regions with established H4K20 modifications (e.g., certain promoters, enhancers, or heterochromatic regions depending on the specific modification).

• Input chromatin: Always prepare an input sample (1-5% of starting chromatin) to normalize ChIP signals and account for differences in DNA abundance.

• Technical positive control: Include an antibody against a highly abundant histone modification (e.g., H3K4me3 at active promoters) as a technical control for the ChIP procedure.

Negative controls:

• IgG control: Perform parallel ChIP with the same amount of non-specific IgG from the same species as the HIST1H4A antibody (rabbit IgG) .

• Genomic regions lacking modification: Design qPCR primers for regions known to lack H4K20 modifications (e.g., certain housekeeping genes or silent genomic regions).

• Peptide competition: Perform ChIP with antibody pre-incubated with the immunizing peptide to demonstrate specificity.

• Isotype control: Use an antibody of the same isotype that targets an irrelevant protein to control for non-specific binding .

How should ChIP-seq data generated with HIST1H4A (Ab-20) Antibody be processed and analyzed?

ChIP-seq data analysis for HIST1H4A (Ab-20) Antibody requires a systematic bioinformatic approach:

• Quality control and preprocessing:

  • Assess sequence quality using FastQC.

  • Trim low-quality bases and adapter sequences.

  • Align reads to the reference genome using Bowtie2 or BWA.

  • Remove PCR duplicates and filter for uniquely mapped reads.

• Peak calling:

  • Use MACS2 with appropriate parameters for histone modifications (--broad for broad peaks).

  • Set FDR threshold (<0.05) and fold enrichment (>2) criteria.

  • Generate normalized bigWig files for visualization.

• Differential binding analysis:

  • Compare peak sets between conditions using DiffBind or similar tools.

  • Normalize for sequencing depth and input control.

  • Apply appropriate statistical methods (DESeq2, edgeR) for differential enrichment analysis.

• Genomic annotation and visualization:

  • Annotate peaks relative to genomic features using HOMER or ChIPseeker.

  • Generate average profile plots and heatmaps using deepTools.

  • Visualize individual loci using genome browsers like IGV or UCSC.

• Integration with other data types:

  • Correlate H4K20 modification patterns with gene expression (RNA-seq).

  • Integrate with other histone modification data to identify combinatorial patterns.

  • Perform motif enrichment analysis to identify associated transcription factors .

What methodological approaches can distinguish between mono-, di-, and tri-methylation of H4K20 when using HIST1H4A antibodies?

Distinguishing between different methylation states of H4K20 requires specific methodological considerations:

• Modification-specific antibodies:

  • Use antibodies specifically targeting H4K20me1, H4K20me2, or H4K20me3 rather than the general HIST1H4A (Ab-20) Antibody.

  • Validate specificity through peptide competition assays using modified and unmodified peptides.

  • Compare ChIP-seq profiles to established patterns (H4K20me1 at active promoters, H4K20me3 at heterochromatin).

• Mass spectrometry approaches:

  • Perform immunoprecipitation using HIST1H4A (Ab-20) Antibody.

  • Analyze precipitated histones by targeted mass spectrometry.

  • Quantify relative abundance of different methylation states at K20.

• Sequential ChIP:

  • Perform first ChIP with HIST1H4A (Ab-20) Antibody.

  • Split the precipitated material and perform second ChIP with modification-specific antibodies.

  • This approach can reveal the distribution of different methylation states within the population of K20-modified histones.

• Immunofluorescence with specific antibodies:

  • Perform multicolor immunofluorescence using antibodies against different methylation states.

  • Analyze co-localization patterns to understand spatial distribution of modifications .

How can HIST1H4A (Ab-20) Antibody data be integrated with other epigenomic datasets to gain insights into chromatin regulation?

Integrative analysis provides comprehensive understanding of epigenetic regulation:

• Multi-omics integration framework:

  • Combine HIST1H4A ChIP-seq data with:

    • Other histone modifications (H3K4me3, H3K27me3, H3K9ac, etc.)

    • DNA methylation data (WGBS, RRBS)

    • Chromatin accessibility data (ATAC-seq, DNase-seq)

    • Transcription factor binding (ChIP-seq)

    • Gene expression (RNA-seq)

    • 3D genome organization (Hi-C, ChIA-PET)

• Computational integration methods:

  • Use dimensionality reduction techniques (PCA, t-SNE, UMAP) to visualize relationships.

  • Apply clustering algorithms to identify chromatin states (ChromHMM, Segway).

  • Implement correlation analyses to identify coordinated changes across datasets.

  • Employ machine learning approaches to predict functional outcomes from epigenetic patterns.

• Pathway and network analysis:

  • Map H4K20 modification patterns to gene regulatory networks.

  • Perform Gene Ontology and pathway enrichment analysis on genes associated with specific modification patterns.

  • Construct protein-protein interaction networks centered on writers, readers, and erasers of H4K20 modifications.

• Visualization strategies:

  • Create multi-track genome browser views showing aligned datasets.

  • Generate correlation heatmaps across different epigenetic marks.

  • Develop circular plots (Circos) to visualize long-range interactions in context of H4K20 modifications .

How can HIST1H4A (Ab-20) Antibody be applied in single-cell epigenomic studies?

Adapting HIST1H4A (Ab-20) Antibody for single-cell approaches requires specialized methodologies:

• Single-cell CUT&Tag or CUT&RUN:

  • Use HIST1H4A (Ab-20) Antibody to target H4K20 modifications in single cells.

  • Implement microfluidic or plate-based single-cell isolation.

  • Add cell-specific barcodes during library preparation.

  • Analyze cell-to-cell variation in H4K20 modification patterns.

• Single-cell ChIP-seq adaptations:

  • Develop micro-ChIP protocols using HIST1H4A (Ab-20) Antibody with reduced cell numbers.

  • Implement carrier ChIP approaches to improve efficiency with limited material.

  • Utilize droplet-based or microwell technologies for single-cell processing.

• Multi-modal single-cell profiling:

  • Combine immunofluorescence using HIST1H4A (Ab-20) Antibody with single-cell RNA-seq.

  • Implement CITE-seq-like approaches to simultaneously measure histone modifications and transcriptomes.

  • Correlate H4K20 modification states with transcriptional heterogeneity at single-cell resolution.

• Spatial epigenomics:

  • Apply HIST1H4A (Ab-20) Antibody in spatial technologies like Visium or MERFISH.

  • Map H4K20 modification patterns in tissue context with spatial resolution.

  • Correlate spatial patterns with tissue architecture and cell type distribution .

What considerations are important when applying HIST1H4A (Ab-20) Antibody in 3D chromatin organization studies?

Investigating 3D chromatin architecture in relation to H4K20 modifications requires specialized approaches:

• ChIP-seq integration with Hi-C data:

  • Perform ChIP-seq using HIST1H4A (Ab-20) Antibody and Hi-C on the same biological samples.

  • Analyze enrichment of H4K20 modifications at topologically associating domain (TAD) boundaries.

  • Investigate correlation between modification patterns and chromatin interaction frequencies.

• ChIA-PET or HiChIP methodologies:

  • Adapt protocols to use HIST1H4A (Ab-20) Antibody for chromatin interaction analysis.

  • Identify chromatin interactions mediated by regions enriched for H4K20 modifications.

  • Compare interaction profiles with those generated using architectural proteins like CTCF.

• Super-resolution microscopy:

  • Perform immunofluorescence with HIST1H4A (Ab-20) Antibody using super-resolution techniques (STORM, PALM, STED).

  • Visualize 3D distribution of H4K20 modifications in nuclear space.

  • Correlate modification patterns with chromatin compartments and nuclear landmarks.

• Genomic editing of H4K20 regulatory enzymes:

  • Generate cell lines with mutations in enzymes that write, read, or erase H4K20 modifications.

  • Analyze changes in 3D genome organization using Hi-C or microscopy.

  • Use HIST1H4A (Ab-20) Antibody to track resulting changes in modification patterns .

What are the detailed specifications of commercially available HIST1H4A (Ab-20) Antibodies?

Below is a comparative table of HIST1H4A (Ab-20) Antibody specifications from different suppliers:

This information helps researchers select the appropriate antibody for their specific experimental requirements and ensure consistent results across studies .

How should HIST1H4A (Ab-20) Antibody be stored and handled to maintain optimal activity?

Proper storage and handling are crucial for maintaining antibody performance:

• Storage conditions:

  • Store antibody at +4°C for short-term use (up to 1 week) .

  • For long-term storage, aliquot and store at -20°C or -80°C to avoid repeated freeze-thaw cycles .

  • Keep in the buffer supplied by the manufacturer, typically containing 50% glycerol and PBS at pH 7.4 .

  • Protect from light, especially if conjugated to fluorophores.

• Aliquoting best practices:

  • Divide the stock solution into small, single-use aliquots (10-20μL).

  • Use sterile, low-protein binding tubes.

  • Minimize time at room temperature during aliquoting.

  • Record date of aliquoting and track freeze-thaw cycles.

• Thawing and handling:

  • Thaw aliquots at 4°C or on ice, never at room temperature.

  • Centrifuge briefly after thawing to collect contents at the bottom of the tube.

  • Mix gently by finger-tapping or gentle pipetting, avoid vortexing.

  • Keep on ice during experimental setup.

• Stability considerations:

  • With each freeze-thaw cycle, antibody can lose approximately half of its binding activity .

  • Working solutions (diluted antibody) should be prepared fresh and used within 24 hours.

  • Monitor performance over time through consistent positive controls .

What quality control measures should be implemented when using a new lot of HIST1H4A (Ab-20) Antibody?

Implementing robust quality control measures helps ensure consistent experimental results:

• Lot-to-lot comparison:

  • Perform side-by-side validation of old and new antibody lots.

  • Compare signal-to-noise ratios in your specific application.

  • Document differences in optimal working dilutions or conditions.

• Standard curve analysis:

  • Generate standard curves using recombinant or purified HIST1H4A protein.

  • Compare detection limits, dynamic range, and EC50 values between lots.

  • Adjust working concentrations based on relative affinity if necessary.

• Application-specific validation:

  • For ChIP: Compare enrichment profiles at known target regions.

  • For IF/IHC: Compare staining patterns and signal intensities.

  • For ELISA: Compare standard curves and detection thresholds.

• Documentation and record-keeping:

  • Maintain detailed records of lot numbers, reception dates, and validation results.

  • Document specific conditions that work optimally for each lot.

  • Consider implementing a laboratory information management system (LIMS) for tracking antibody performance over time .