HIST1H4A (Ab-31) Antibody

What is HIST1H4A and what specific epitope does the HIST1H4A (Ab-31) Antibody recognize?

HIST1H4A is one of several genes encoding histone H4, a core component of the nucleosome. The nucleosome is the fundamental repeating unit of chromatin, consisting of approximately 146 base pairs of DNA wrapped around an octamer of histones (two copies each of H2A, H2B, H3, and H4).

The HIST1H4A (Ab-31) Antibody specifically recognizes the region around lysine 31 (Lys31) in human histone H4. This antibody is generated using a peptide sequence derived from human histone H4 containing this specific residue as the immunogen. The antibody is particularly useful for detecting post-translational modifications at this site .

Histone H4 is highly conserved across species and plays crucial roles in chromatin packaging, gene regulation, and epigenetic processes. Its modifications, including acetylation, methylation, and phosphorylation, are key regulatory mechanisms in gene expression and cellular processes.

Which research applications is the HIST1H4A (Ab-31) Antibody validated for?

The HIST1H4A (Ab-31) Antibody has been validated for multiple research applications, providing versatility for epigenetic studies:

This polyclonal antibody raised in rabbit demonstrates specificity for human samples. While cross-reactivity with other species may occur due to the high conservation of histone sequences, specific validation for other species should be conducted by researchers .

How does HIST1H4A (Ab-31) Antibody differ from antibodies targeting other histone H4 modifications?

HIST1H4A (Ab-31) Antibody specifically targets the region around lysine 31 of histone H4, distinguishing it from antibodies targeting other modifications:

When selecting a histone H4 antibody, researchers should consider which specific modification they aim to study, as each modification has distinct functions in chromatin regulation. Using multiple antibodies targeting different modifications can provide a more comprehensive understanding of the epigenetic landscape.

What are appropriate positive controls for validating HIST1H4A (Ab-31) Antibody in experiments?

Proper validation of HIST1H4A (Ab-31) Antibody requires appropriate positive controls:

• Cell line lysates: Human cell lines known to express histone H4 abundantly, such as HeLa, HEK293, or Jurkat cells, serve as excellent positive controls for Western blot applications.

• Recombinant histone H4: Purified recombinant histone H4 protein can be used as a standard in Western blots or ELISA to confirm antibody specificity.

• FFPE human tissue sections: For IHC applications, human tissue sections containing cell types with known histone H4 expression patterns can serve as positive controls.

• Modification-enriched samples: For studying specific acetylation at Lys31, samples treated with histone deacetylase inhibitors (e.g., trichostatin A, sodium butyrate) can enhance acetylation levels, providing enriched positive controls .

• Peptide competition: Using the immunizing peptide to block antibody binding can confirm specificity by demonstrating signal reduction.

Histone H4 is universally expressed with a molecular weight of approximately 11 kDa , making it readily detectable in most cellular contexts when using appropriate techniques.

How can HIST1H4A (Ab-31) Antibody be optimized for chromatin immunoprecipitation (ChIP) experiments?

Optimizing ChIP protocols with HIST1H4A (Ab-31) Antibody requires careful consideration of several parameters:

Cross-linking and Sonication:

• Use 1% formaldehyde for 10 minutes at room temperature for optimal cross-linking

• Sonicate chromatin to fragments of 200-500 bp (verify by agarose gel electrophoresis)

• Ensure consistent sonication across samples to avoid bias

Immunoprecipitation Conditions:

• Pre-clear chromatin with protein A/G beads to reduce non-specific binding

• Use 2-5 μg antibody per ChIP reaction

• Perform overnight incubation at 4°C with rotation

• Include input controls (10% of starting material) and IgG controls

Washing and Elution:

• Implement stringent washing steps to reduce background

• Optimize buffer compositions based on target specificity

• Consider sequential ChIP for studying co-occurrence with other modifications

Data Analysis Considerations:

• Normalize to input DNA

• Use appropriate normalization methods for quantitative comparisons

• Consider the genomic distribution patterns of histone H4 modifications

A common challenge in histone ChIP experiments is ensuring the specificity of the antibody for the particular modification of interest. Cross-reactivity with similar modifications can occur, so validation with peptide competition assays or using cells with known modification patterns is essential for reliable results .

What are the key considerations when investigating histone H4 acetylation patterns in relation to transcriptional regulation?

Investigating histone H4 acetylation patterns in transcriptional regulation requires a multifaceted approach:

Genome-wide Distribution Analysis:

• ChIP-seq using HIST1H4A (Ab-31) Antibody can reveal genome-wide distribution of H4K31 acetylation

• Compare with transcriptome data (RNA-seq) to correlate modification with gene expression

• Analyze distribution relative to transcription start sites, enhancers, and other regulatory elements

Temporal Dynamics:

• Study acetylation changes during different cellular processes or developmental stages

• Time-course experiments can reveal dynamic regulation of H4K31ac during transcriptional responses

Integration with Other Histone Modifications:

• Compare H4K31ac patterns with other histone modifications (H3K4me3, H3K27ac, etc.)

• The presence of H3K4me3 at promoters is often associated with active transcription, where it is typically flanked by lower H3K4 methylation states

• H4K31ac may function in conjunction with these established modifications

Enzyme Interactions:

• Identify histone acetyltransferases (HATs) and deacetylases (HDACs) that regulate H4K31 acetylation

• Use inhibitors of these enzymes to modulate acetylation levels and observe effects on transcription

Research has demonstrated that histone modifications often work in combination, creating a "histone code" that influences chromatin structure and gene expression. For instance, H3K4me3 is phenomenologically and biochemically associated with active promoters, where it is flanked by lower H3K4 methylation states . Understanding how H4K31ac fits into this complex regulatory network requires comprehensive analysis alongside other modifications.

How can HIST1H4A (Ab-31) Antibody be employed in dual immunofluorescence studies to investigate co-localization with other histone modifications?

Dual immunofluorescence studies offer valuable insights into the spatial relationships between different histone modifications:

Protocol Optimization:

• Fixation: Use 4% paraformaldehyde for 15 minutes at room temperature

• Permeabilization: 0.2% Triton X-100 for 10 minutes

• Blocking: 5% BSA or 10% normal serum from the species of secondary antibody

• Primary antibody incubation: Use HIST1H4A (Ab-31) Antibody at 1:50-1:200 dilution

• Secondary antibody selection: Choose spectrally distinct fluorophores for each primary antibody

• Nuclear counterstain: DAPI for nuclear identification

Co-localization Analysis:

• Calculate Pearson's or Mander's coefficients to quantify co-localization

• Perform pixel intensity correlation analysis along nuclear regions

• Consider 3D confocal microscopy for volumetric analysis of co-localization

Experimental Design Considerations:

• Include single-stain controls to assess bleed-through

• Use absorption controls to verify antibody specificity

• Include biological controls with known modification patterns

Technical Challenges and Solutions:

• Challenge: Cross-reactivity between antibodies

  • Solution: Use antibodies raised in different host species

• Challenge: Signal-to-noise ratio

  • Solution: Optimize antibody concentration and implement signal amplification methods

• Challenge: Simultaneous detection of multiple modifications

  • Solution: Consider sequential immunostaining or spectral unmixing techniques

When investigating co-localization of H4K31ac with other modifications, such as H3K4me3, researchers can gain insights into the functional relationships between different epigenetic marks and their collective impact on chromatin organization and gene expression .

What are common issues encountered when using HIST1H4A (Ab-31) Antibody in Western blotting and how can they be resolved?

Western blotting with histone antibodies presents unique challenges that require specific troubleshooting strategies:

Specific Recommendations for HIST1H4A (Ab-31) Antibody:

• Protein Extraction: Use specialized histone extraction protocols (e.g., acid extraction with 0.2N HCl) to enrich for histones.

• Gel Electrophoresis: Use high percentage (15-18%) SDS-PAGE gels to effectively resolve the low molecular weight (11 kDa) histone H4 .

• Transfer Optimization: Implement specialized transfer conditions for small proteins:

  • Use PVDF membrane (0.2 μm pore size)

  • Consider semi-dry transfer or use transfer buffers with reduced methanol

  • Transfer at lower voltage for longer time

• Blocking: Use 5% non-fat dry milk or BSA in TBST for 1-2 hours at room temperature.

• Antibody Incubation: Dilute primary antibody appropriately and incubate overnight at 4°C with gentle agitation.

• Visualization: Consider using enhanced chemiluminescence (ECL) detection methods with extended exposure times if necessary.

How should researchers address specificity concerns when working with histone modification antibodies like HIST1H4A (Ab-31)?

Antibody specificity is critical for histone modification research and requires rigorous validation:

Experimental Validation Approaches:

• Peptide Competition Assays:

  • Pre-incubate antibody with immunizing peptide

  • Compare signal with and without peptide competition

  • Specific binding should be significantly reduced with peptide competition

• Modified vs. Unmodified Peptide Arrays:

  • Test antibody against arrays containing modified and unmodified histone peptides

  • Quantify relative binding to assess specificity for the target modification

• Genetic Validation:

  • Use cells with genetic alterations in histone modification enzymes

  • Compare signal in wild-type vs. enzyme-deficient cells

  • Signal should be reduced in cells lacking the specific modification

• Mass Spectrometry Correlation:

  • Compare ChIP-seq or immunostaining results with quantitative mass spectrometry data

  • Verify that antibody enrichment correlates with modification abundance

Addressing Cross-Reactivity:

The high sequence similarity between histone variants and the presence of similar modification sites can lead to cross-reactivity. For instance, the antibody may recognize similar acetylation sites on histone H4 beyond K31. Researchers should be aware that histone H4 has multiple acetylation sites, including lysines 5, 8, 12, 16, and 31, which may share sequence context .

To address this:

• Test against a panel of modified peptides covering various acetylation sites

• Include appropriate controls in each experiment

• Consider using specialized methods like ICeChIP (Internal Standard Calibrated ChIP) for quantitative assessment of antibody specificity

How should researchers interpret ChIP-seq data generated using HIST1H4A (Ab-31) Antibody in relation to gene regulation?

Interpreting ChIP-seq data for H4K31ac requires careful analysis and integration with other genomic data:

Analysis Pipeline:

• Quality Control and Preprocessing:

  • Assess sequencing quality (FastQC)

  • Trim adaptors and low-quality reads

  • Align to reference genome (e.g., hg38 for human samples)

  • Remove PCR duplicates and normalize for sequencing depth

• Peak Calling and Annotation:

  • Use appropriate peak callers (MACS2, HOMER) optimized for histone modifications

  • Annotate peaks relative to genomic features (promoters, enhancers, gene bodies)

  • Calculate enrichment at transcription start sites (TSS) and other regulatory regions

• Integration with Gene Expression Data:

  • Correlate H4K31ac enrichment with RNA-seq or microarray data

  • Perform gene ontology analysis of genes associated with H4K31ac

  • Consider time-course analyses to identify dynamic relationships

Interpretation Framework:

Comparative Analysis:

Researchers should compare H4K31ac patterns with established histone modifications:

• H3K4me3 marks active promoters

• H3K4me1 typically marks enhancers and flanks promoters (5-20% global abundance)

• H3K4me2 is associated with tissue-specific transcription factor binding sites (1-4% global abundance)

This comparative approach can help position H4K31ac within the broader context of the histone code and gene regulation mechanisms.

What statistical methods are most appropriate for analyzing co-localization between H4K31ac and other histone modifications?

Genome-wide Co-localization Analysis:

• Peak Overlap Analysis:

  • Calculate the significance of peak overlaps using permutation tests

  • Apply methods like LOLA (Locus Overlap Analysis) or GenometriCorr

  • Normalize for genomic distribution biases

• Correlation Analysis:

  • Calculate Pearson or Spearman correlation of signal intensities

  • Perform these calculations within specific genomic contexts (promoters, enhancers)

  • Generate correlation heatmaps for multiple histone modifications

• Enrichment Analysis:

  • Calculate observed/expected ratios for co-occurrence

  • Implement windowing approaches to assess proximity relationships

  • Use tools like ChromHMM to identify combinatorial chromatin states

Imaging-based Co-localization Analysis:

For microscopy data:

• Pearson's correlation coefficient: Measures linear correlation between intensity values

• Mander's overlap coefficient: Quantifies the proportion of overlapping signals

• Object-based methods: Assess spatial relationships between identified structures

Statistical Considerations:

Data Visualization:

• Generate metaplots centered on genomic features of interest

• Create heatmaps showing signal distributions across genes

• Use browser tracks for specific loci visualization

• Implement 2D density plots for pairwise modification comparisons

These statistical approaches should be tailored to the specific biological questions and experimental design, keeping in mind that correlative relationships may suggest, but do not prove, functional relationships between histone modifications.

How can researchers differentiate between functional significance and coincidental co-occurrence when studying histone H4 modifications?

Distinguishing functional relationships from coincidental associations requires multiple lines of evidence:

Experimental Approaches:

• Perturbation Studies:

  • Inhibit or knock down enzymes responsible for specific modifications

  • Observe effects on other modifications and functional outcomes

  • Use site-specific histone mutants (when possible) to directly test function

• Time-course Experiments:

  • Monitor temporal dynamics of different modifications

  • Establish order of appearance/disappearance

  • Identify potential causal relationships

• Single-cell Analysis:

  • Examine modification heterogeneity within cell populations

  • Correlate with functional states at single-cell level

  • Look for coordinated changes across modifications

Analytical Frameworks:

• Bayesian Network Analysis:

  • Infer directional relationships between modifications

  • Account for confounding variables

  • Model potential causal structures

• Motif Analysis:

  • Identify enriched DNA sequence motifs associated with modification patterns

  • Connect to transcription factor binding sites

  • Link to known regulatory elements

• Evolutionary Conservation:

  • Compare modification patterns across species

  • Functionally significant relationships often show evolutionary conservation

  • Analyze syntenic regions for conservation of modification patterns

Contextual Considerations:

Researchers should consider:

• Cell type-specific effects

• Developmental stage-specific patterns

• Disease state alterations

• Interaction with non-histone proteins