HIST1H4A (Ab-79) Antibody

What is HIST1H4A (Ab-79) Polyclonal Antibody and what does it target?

HIST1H4A (Ab-79) Polyclonal Antibody is a primary antibody that specifically recognizes the region around lysine 79 of human Histone H4 protein. It is generated in rabbits using a peptide sequence derived from Human Histone H4 as the immunogen, particularly targeting the region surrounding the lysine 79 residue. The antibody recognizes the human (Homo sapiens) HIST1H4A protein, which has the accession number P62805 . Histone H4 is a core component of the nucleosome, the fundamental unit of chromatin structure, and is involved in various cellular processes including gene regulation, DNA repair, and chromatin assembly.

What applications has this antibody been validated for?

The HIST1H4A (Ab-79) Polyclonal Antibody has been specifically validated for three primary research applications:

• ELISA (Enzyme-Linked Immunosorbent Assay): For quantitative detection of HIST1H4A protein in solution-based assays

• IHC (Immunohistochemistry): For detection of the protein in tissue sections

• ChIP (Chromatin Immunoprecipitation): For investigating protein-DNA interactions and histone modifications

These validations make it a versatile tool for researchers investigating histone H4 biology across different experimental platforms. Each application requires specific optimization protocols for maximum specificity and sensitivity.

What are the various synonyms and nomenclature associated with the target protein?

The target protein has numerous synonyms in the scientific literature, which can create confusion when searching databases or comparing research findings. The comprehensive list includes: dJ160A22.1, dJ160A22.2, dJ221C16.1, dJ221C16.9, FO108, H4, H4.k, H4/a, H4/b, H4/c, H4/d, H4/e, H4/g, H4/h, H4/I, H4/j, H4/k, H4/m, H4/n, H4/p, H4_HUMAN, H4F2, H4F2iii, H4F2iv, H4FA, H4FB, H4FC, H4FD, H4FE, H4FG, H4FH, H4FI, H4FJ, H4FK, H4FM, H4FN, H4M, HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4L, HIST2H4, HIST2H4A, Hist4h4, and various "Histone" designations .

This extensive nomenclature reflects the complexity of histone gene clusters and the conservation of histone proteins across different genomic loci. Researchers should be aware of these alternative names when conducting literature searches or comparing experimental results across different studies.

What controls should be included when using this antibody for ChIP experiments?

When designing ChIP experiments with the HIST1H4A (Ab-79) antibody, several essential controls should be implemented:

• Input Control: Set aside a small portion (5-10%) of the chromatin before immunoprecipitation to normalize for differences in chromatin preparation and DNA amount.

• Isotype Control: Use a non-specific rabbit IgG antibody under identical conditions to assess non-specific binding.

• Positive Control Loci: Include primers for genomic regions known to be enriched for H4 modifications, particularly active regions of the genome where H4 K91 acetylation has been shown to be significantly enriched .

• Negative Control Loci: Include primers for regions with low or absent H4 modifications, such as silent heterochromatic regions. Research has shown that telomeres and the HMR locus typically show low levels of H4 K91 acetylation and could serve as appropriate negative controls .

• Specificity Control: When possible, include samples from cells expressing the H4 K91A mutation to demonstrate antibody specificity, as ChIP signals should be absent or significantly reduced in these samples .

How should sample preparation be optimized for detecting HIST1H4A using this antibody?

Optimal sample preparation is critical for successful detection of HIST1H4A using this antibody:

• For IHC applications:

  • Fixation: 10% neutral buffered formalin for 24-48 hours

  • Antigen retrieval: Heat-induced epitope retrieval using citrate buffer (pH 6.0) is recommended

  • Blocking: Use 5-10% normal serum from the same species as the secondary antibody

  • Antibody dilution: Determine optimal dilution through titration experiments (typical range: 1:100-1:500)

• For ChIP applications:

  • Crosslinking: 1% formaldehyde for 10 minutes at room temperature

  • Sonication: Optimize to achieve DNA fragments of 200-500 bp

  • Chromatin amount: 25-100 μg of chromatin per immunoprecipitation

  • Antibody amount: 2-5 μg per immunoprecipitation

  • Incubation: Overnight at 4°C with rotation

• For ELISA applications:

  • Coating concentration: 1-10 μg/ml of target protein

  • Blocking: 1-5% BSA or non-fat dry milk

  • Antibody dilution: Determine optimal dilution through titration (typical range: 1:1000-1:5000)

  • Detection: HRP-conjugated secondary antibody with appropriate substrate

These parameters should be optimized for each specific experimental system to ensure maximum sensitivity and specificity.

How can this antibody be used to study the relationship between histone H4 modifications and chromatin structure?

The HIST1H4A (Ab-79) antibody can be instrumental in investigating the complex relationship between histone H4 modifications and chromatin structure through several advanced approaches:

• Sequential ChIP (Re-ChIP): This technique allows for the identification of nucleosomes containing multiple histone modifications by performing successive immunoprecipitations. Researchers can use the HIST1H4A (Ab-79) antibody in combination with antibodies against other histone modifications to determine co-occurrence patterns.

• ChIP-seq Analysis: By combining ChIP with next-generation sequencing, researchers can generate genome-wide maps of H4 modifications. This approach has revealed that H4 K91 acetylation, a modification in the globular domain of histone H4, is significantly enriched in active regions of the genome while showing low levels at telomeres and the HMR locus .

• Hydroxyapatite Chromatography: This technique can be used to assess histone octamer stability. Research has shown that modifications in the histone H4 globular domain, particularly at lysine 91, affect the stability of histone octamers and the interaction between H2A/H2B dimers and H3/H4 tetramers. This antibody can help investigate similar effects at lysine 79 .

• Chromatin Assembly Assays: Since H4 modifications are involved in chromatin assembly, this antibody can be used to study the temporal dynamics of H4 modifications during this process, particularly in conjunction with type B histone acetyltransferases (HATs) .

What approaches can be used to validate antibody specificity for HIST1H4A (Ab-79)?

Validating antibody specificity is crucial for reliable research outcomes. For the HIST1H4A (Ab-79) antibody, several validation approaches are recommended:

• Peptide Competition Assay: Pre-incubate the antibody with excess immunizing peptide (the specific peptide sequence around site of Lys 79 derived from Human Histone H4) before application to the sample. A true specific signal should be blocked or significantly reduced.

• Dot Blot Analysis: Test the antibody against modified and unmodified peptides encompassing several sites of H4 acetylation to confirm site-specific recognition. This approach has been effectively used for similar antibodies targeting H4 modifications .

• Genetic Controls: Use samples from cells expressing H4 K79A mutations (similar to the H4 K91A approach described in the literature) to demonstrate that the antibody fails to detect the target when the specific lysine residue is altered .

• Cross-Reactivity Assessment: Pre-incubate the antibody with lysate from cells containing a mutation at the target site (e.g., H4 K79A) to block any potential cross-reactivity with other histone modifications before using in assays such as ChIP .

• Western Blot Analysis: Perform western blot on recombinant H4 protein expressed in E. coli with and without the target modification to confirm specificity for the modified form.

How can researchers distinguish between different histone H4 variants when using this antibody?

Distinguishing between histone H4 variants poses a significant challenge due to their high sequence similarity. When using the HIST1H4A (Ab-79) antibody, consider these approaches:

• Combinatorial Immunoprecipitation: Combine the HIST1H4A antibody with antibodies against specific histone chaperones known to preferentially interact with particular H4 variants.

• Mass Spectrometry Validation: After immunoprecipitation with the HIST1H4A antibody, analyze the precipitated proteins by mass spectrometry to identify specific histone variants based on unique peptide signatures.

• Sequential ChIP with Variant-Specific Antibodies: If available, use sequential ChIP with antibodies that can distinguish between specific histone variants based on their unique regions.

• Expression System Controls: Use cells engineered to express only certain H4 variants to establish baseline reactivity patterns for the antibody.

• Bioinformatic Analysis of ChIP-seq Data: Advanced computational approaches can help distinguish binding patterns characteristic of different histone variants when combined with genomic features and other epigenetic marks.

What are common causes of false positive or negative results when using this antibody?

Common causes of false results when using the HIST1H4A (Ab-79) antibody include:

False Positives:

• Insufficient blocking leading to non-specific binding

• Cross-reactivity with similar histone modifications

• Excessively high antibody concentration

• Contamination during sample preparation

• Non-specific binding to denatured or exposed epitopes after aggressive antigen retrieval

False Negatives:

• Inefficient antigen retrieval (particularly important for fixed tissues)

• Epitope masking due to protein-protein interactions

• Low abundance of the target modification

• Degradation of the target protein during sample preparation

• Improper antibody storage leading to reduced activity

To minimize these issues, researchers should:

• Validate the antibody in their specific experimental system using the approaches described in section 3.2

• Include appropriate positive and negative controls

• Optimize experimental conditions through careful titration experiments

• Store the antibody according to manufacturer recommendations

How should researchers interpret ChIP data in the context of histone H4 modification patterns?

Interpretation of ChIP data for histone H4 modifications requires consideration of several factors:

• Genomic Context: H4 modifications often show distinct patterns in different genomic regions. For example, H4 K91 acetylation is enriched in active genomic regions while showing low levels at telomeres and silent loci .

• Temporal Dynamics: Consider the cell cycle stage and developmental timepoints, as histone modifications can change dynamically during these processes.

• Co-occurrence with Other Marks: Interpret H4 modification data in the context of other histone modifications, as they often function in combination. For instance, H4 K91 acetylation appears to be mechanistically distinct from modifications dependent on HAT1 .

• Chromatin Structure Effects: Some H4 modifications, particularly those in the globular domain, can affect histone octamer stability. The H4 K91A mutation, for example, destabilizes the interaction between H2A/H2B dimers and H3/H4 tetramers .

• Functional Effects: Connect modification patterns to functional outcomes like transcriptional activity, DNA repair, or chromatin assembly. Mutations that alter key residues in H4 can confer phenotypes consistent with defects in these processes .

What data normalization and statistical approaches are recommended for quantitative analysis using this antibody?

For rigorous quantitative analysis of data generated using the HIST1H4A (Ab-79) antibody:

• ChIP-qPCR Normalization:

  • Percent of input method: Calculate enrichment as a percentage of input chromatin

  • Fold enrichment over IgG control: Compare specific antibody signal to non-specific IgG signal

  • Normalization to a reference gene: Express target enrichment relative to a consistently expressed reference gene

• ChIP-seq Analysis:

  • Use spike-in controls (e.g., Drosophila chromatin) for absolute quantification

  • Employ RPKM (Reads Per Kilobase Million) for comparing enrichment across samples

  • Apply appropriate peak calling algorithms (e.g., MACS2) with stringent FDR control

• IHC Quantification:

  • Use digital image analysis with standardized acquisition settings

  • Quantify staining intensity using H-score or Allred scoring systems

  • Include tissue microarrays with control samples for batch normalization

• Statistical Approaches:

  • For ChIP-qPCR: Use paired t-tests or ANOVA for comparing enrichment across conditions

  • For ChIP-seq: Apply DESeq2 or edgeR for differential binding analysis

  • For IHC: Use non-parametric tests (Mann-Whitney U or Kruskal-Wallis) for scoring comparisons

• Multiple Testing Correction:

  • Apply Benjamini-Hochberg procedure for controlling false discovery rate in genome-wide analyses

  • Use Bonferroni correction for family-wise error rate control in targeted analyses

How can this antibody be integrated into multi-omics experimental workflows?

The HIST1H4A (Ab-79) antibody can be effectively incorporated into integrated multi-omics workflows:

• ChIP-seq + RNA-seq Integration: Correlate H4 modification patterns with transcriptional outputs to establish functional relationships between specific modifications and gene expression.

• ChIP-MS (Chromatin Immunoprecipitation followed by Mass Spectrometry): Identify proteins that interact with regions containing modified H4 to elucidate the readers and effectors of specific histone modifications.

• CUT&RUN or CUT&Tag + ATAC-seq: Combine precise mapping of H4 modifications using CUT&RUN or CUT&Tag (which require less sample input than traditional ChIP) with accessibility data from ATAC-seq to understand how these modifications affect chromatin structure.

• HiChIP (Protein-Centric Chromatin Conformation): Integrate this antibody into HiChIP protocols to investigate how H4 modifications influence three-dimensional chromatin architecture.

• Single-Cell Multi-Omics: Adapt protocols for use in single-cell epigenomics approaches to investigate cell-to-cell heterogeneity in H4 modification patterns and correlate with other cellular parameters.

What insights can be gained from studying HIST1H4A in autoimmune conditions?

Recent research in autoimmunity suggests that histone proteins, including H4, may play important roles as autoantigens:

• Autoantibody Profiling: Studies have established comprehensive catalogs of autoantibody profiles in various conditions, with elevated levels of autoantibodies observed in diseases such as COVID-19, systemic sclerosis (SSc), and systemic lupus erythematosus (SLE) .

• Disease-Specific Biomarkers: Certain autoantibodies show disease-specific patterns of elevation and may serve as biomarkers. While the HIST1H4A antibody itself is a research tool rather than an autoantibody, it can be used to study how histone H4 modifications might affect the immunogenicity of chromatin in autoimmune conditions.

• Temporal Dynamics: Longitudinal studies of autoantibodies, such as those conducted in COVID-19 patients with samples collected at different time points after symptom onset, provide insights into the temporal dynamics of autoimmune responses . Similar approaches could be applied to study histone H4 modifications during disease progression.

• Threshold Determination: Establishing thresholds for positivity based on Z-scores calculated from healthy control distributions (e.g., Z-scores > 4) can help distinguish pathological from normal levels of autoantibodies or histone modifications .

• Clinical Correlations: While some specific autoantibodies (e.g., anti-BCORP1, anti-KAT2A) have been investigated for associations with clinical outcomes in conditions like COVID-19 , similar approaches could be applied to study correlations between histone H4 modifications and disease severity or progression.